Biomarkers for efficacy of prophylactic treatments against stress-induced affective disorders

ABSTRACT

Biomarkers for assessing the efficacy of prophylactic treatments of stress-induced affective disorders are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 62/583,734 filed on Nov. 9, 2017, which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersOD017908 and HD007430 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to biomarkers for assessing the efficacyof prophylactic treatments of stress-induced affective disorders such aspost-traumatic stress disorder (PTSD).

BACKGROUND OF THE INVENTION

Stress is a common risk factor for psychiatric disorders such as majordepressive disorder (MDD), posttraumatic stress disorder (PTSD), bipolardepression, and anxiety disorders. PTSD is an illness characterized bypersistent, vivid re-experiencing of a traumatic event, hyperarousal,and avoidance of stimuli associated with the trauma (Charney et al.,1993, Psychobiologic mechanisms of posttraumatic stress disorder. ArchGen Psychiatry 50:295-305). The National Center for PTSD reports that7-8% of the United States population will experience PTSD at some pointin their lives, and about 8 million adults suffer from the disorder eachyear (National Center for PTSD, 2015). PTSD is often comorbid with otherprevalent psychiatric illnesses such as major depressive disorder (MDD)(28%) and substance use (73%) (Brady et al., 2000, Comorbidity ofpsychiatric disorders and posttraumatic stress disorder. J ClinPsychiatry 61: 22-32).

Ketamine, a non-selective glutamate N-methyl-D-aspartate (NMDA)antagonist, has been shown to have rapid acting antidepressant effectsin treatment-resistant MDD (TRD) patients (Berman et al, 2000). Theantidepressant onset is typically within 2 hours and these effectspersist for 1-2 weeks following a single infusion (Price et al, 2009;Zarate et al, 2006). In addition to having efficacy in MDD, ketamine hastherapeutic effects for PTSD (Feder et al, 2014), suicide ideation(Murrough et al, 2015), bipolar depression (Diazgranados et al, 2010;Zarate et al, 2012), and anxious bipolar depression (Ionescu et al,2015).

Rather than treating symptomatology in psychiatric disorders, a novelapproach has recently been suggested to prevent the induction ofpsychiatric disorders before they develop by administering prophylacticpharmaceuticals. It has been proposed that it may be possible to takeprevention approaches, including pharmacological ones, for PTSD (Howlettand Stein, 2016). One study administered the kappa-opioid receptor (KOR)antagonist nor-binaltorphimine (norBNI) before the fear-potentiatedstartle (FPS) test and found that it attenuated conditioned fear (Knollet al, 2007; Van′t Veer and Carlezon, 2013). More recently, we foundthat a single injection of ketamine (30 mg kg⁻¹) 1 week before astressor prevented stress-induced depressive-like behavior and socialavoidance behavior for up to 4 weeks after injection (Brachman et al,2016). Ketamine was efficacious as a prophylactic in 3 mouse models ofstress, including learned helplessness (LH), social defeat (SD), andchronic corticosterone (CORT) administration. This efficacy wasconfirmed using a LH model in rats (Amat et al, 2016) and a chronic CORTmodel in mice (Soumier et al, 2016). Interestingly, the time window forprophylactic efficacy was limited to approximately 1 week before astressor, but not 1 month or 1 hour before (McGowan et al, 2017).

To date, the mechanisms underlying prophylactic efficacy have yet to beidentified. One potential area of interest is metabolomics, the study ofchemical processes involving metabolites. The metabolome represents acollection of all metabolites, the end products of cellular processes,and therefore, the end results of potential disease or drug treatment;as such, metabolomics has been applied as a tool for biomarker discovery(Johnson et al, 2016). Analyzing metabolite profiles has alreadycontributed to our understanding of depression and antidepressanttreatment in mice, macaques, and humans (Pan et al, 2016; Park et al,2016; Rotroff et al, 2016; Weckmann et al, 2014; Wen et al, 2016).Altered metabolites (e.g. decreased levels of 5-hydroxindoleacitchydroxyindoleacetic acid (5-HIAA)) have been identified in cerebralspinal fluid (Asberg et al, 1976; Pan et al, 2011), urine, and plasmasamples in depressed patients with a markedly elevated risk of suicide(Pan et al, 2017). However, uncovering metabolites that are implicatedin prophylactic efficacy has yet to be explored. Elucidating metabolicchanges in the brain and plasma that are changed with prophylactictreatment may lend insight into the biological mechanisms oflong-lasting resilience enhancement.

Prophylactic pharmacological treatment with ketamine may be able topreempt the induction of stress-induced psychiatric disorders. However,given that up to a third of depression patients do not respond topharmacological therapies, there remains a need for a method to identifyindividuals who are likely to benefit from such interventions.

SUMMARY

The present disclosure provides for a method for treating a subject fora stress-induced affective disorder or stress-induced psychopathology.The method may comprise the following steps: (a) administering or havingadministered a prophylactic treatment to the subject prior to astressor; (b) determining the level of one or more biomarkers in abiological sample obtained from the subject after step (a) and after thestressor; and (c) comparing the level obtained in step (b) with thelevel of the one or more biomarkers in a control sample.

The present disclosure provides for a method for assessing aprophylactic treatment of a subject for a stress-induced affectivedisorder or stress-induced psychopathology. The method may comprise thefollowing steps: (a) determining the level of one or more biomarkers ina biological sample obtained from the subject after a prophylactictreatment and after a stressor; and (b) comparing the level obtained instep (a) with the level of the one or more biomarkers in a controlsample.

The present method may further comprise maintaining a treatment regimeor determining the prophylactic treatment as being effective, when/ifthe level of one or more biomarkers increases or decreases by at leastabout 10%, at least about 15%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 100%,at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.8-fold, atleast 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, atleast 4-fold, at least 4.5-fold, at least 5-fold, from about 30% toabout 3-fold, from about 40% to about 2.5-fold, from about 50% to about2-fold, or from about 60% to about 1.5-fold, compared to its level inthe control sample.

The present method may further comprise adjusting a treatment regime,when/if the level of one or more biomarkers is unchanged, or increasesor decreases by less than about 10%, less than about 15%, less thanabout 20%, less than about 30%, less than about 40%, less than about50%, less than about 60%, less than about 70%, less than about 80%, lessthan about 90%, less than about 100%, less than 1.1-fold, less than1.2-fold, less than 1.3-fold, less than 1.4-fold, less than 1.5-fold,less than 1.6-fold, less than 1.8-fold, less than 2-fold, less than2.5-fold, less than 3-fold, less than 3.5-fold, less than 4-fold, lessthan 4.5-fold, or less than 5-fold, compared to its level in the controlsample.

The biomarkers may be purines, purine metabolites, purine precursors,purine derivatives, pyrimidines, pyrimidine metabolites, pyrimidineprecursors, pyrimidine derivatives, nucleotides, nucleotide metabolites,nucleotide precursors, nucleotide derivatives, neurotransmitters,neurotransmitter metabolites, neurotransmitter precursors,neurotransmitter derivatives, and combinations thereof.

The neurotransmitters may be inhibitory neurotransmitters or excitatoryneurotransmitters. The neurotransmitters may be amino acid-derivedneurotransmitters.

In certain embodiments, the biomarkers are selected from those listed inFIGS. 5C-5Q. In certain embodiments, the biomarkers are selected fromthe group consisting of adenine, adenosine monophosphate (AMP),adenosine triphosphate (ATP), D-ribose 5-phosphate, D-ribulose5-phosphate, guanosine monophosphate, guanosine diphosphate,hypoxanthine, inosine, phosphoribosyl pyrophosphate, and combinationsthereof. In certain embodiments, the biomarkers are selected from thegroup consisting of cytidine monophosphate, dihydrothymine, uridine5-monophosphate, and uridine 5-diphosphate, 5,6-dihydrouridine, andcombinations thereof. In certain embodiments, the biomarkers areselected from those listed in Table 3. In certain embodiments, thebiomarkers are selected from the group consisting of serine, glutamicacid, gamma-aminobuytric acid (GABA), 5-hydroxy-L-tryptophan (5-HTP),O-phosphoethanolamine (PE), N-acetyl-L-tyrosine, and combinationsthereof.

The prophylactic treatment may comprise administering an effectiveamount of a pharmaceutic composition comprising ketamine, a ketamineanalog, or a pharmaceutically acceptable salt, derivative, or metabolitethereof, to the subject prior to the stressor.

The prophylactic treatment may comprise administering an effectiveamount of a pharmaceutic composition comprising an antagonist of theglutamate N-methyl-D-aspartate (NMDA) receptor to a subject prior to astressor. The antagonist of the NMDA receptor may comprise ketamine, aketamine analog, or a pharmaceutically acceptable salt, derivative, ormetabolite thereof.

The prophylactic treatment may comprise administering an effectiveamount of a pharmaceutic composition comprising anα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptoragonist, to a subject prior to a stressor. The AMPA receptor agonist maybe selected from the group consisting of glutamate, AMPA,5-fluorowillardiine, domoic acid, quisqualic acid, and(2R,6R)-hydroxynorketamine, CX546, or a pharmaceutically acceptablesalt, derivative, or metabolite thereof.

The pharmaceutic composition may be administered to the subject about 48hours to about 3 weeks, about 72 hours to about 2 weeks, about 1 week,about 13 days, about 12 days, about 11 days, about 10 days, about 9days, about 8 days, about 7 days, about 6 days, about 5 days, about 4days, about 3 days, about 2 days, or about 1 day, prior to a stressor.

The pharmaceutic composition may be administered to the subject onceprior to a stressor. The pharmaceutical composition may be administeredin a booster series.

The pharmaceutic composition may be administered orally, intravenously,intranasally, or via injection to the subject.

The pharmaceutic composition may comprise norketamine, hydroxyketamines,dehydronorketamine, and/or hydroxynorketamine (HNK). The pharmaceuticcomposition may comprise (2R,6R)-HNK and/or (2S,6S)-HNK.

The biological sample may be a plasma, serum, blood and/or urine sample.

The control sample may be from a healthy subject or a plurality ofhealthy subjects. The control sample may be from a subject who has had aprophylactic treatment without experiencing a stressor, or a pluralityof patients who have had a prophylactic treatment without experiencing astressor. The control sample may be from a subject who has experienced astressor without a prophylactic treatment, or from a plurality ofpatients who have experienced a stressor without a prophylactictreatment.

In one embodiment, the level of the one or more biomarkers is determinedby mass spectrometry (MS). In one embodiment, the level of the one ormore biomarkers is determined by chromatography coupled with MS.

The stress-induced affective disorder may comprise major depressivedisorder (MDD) and/or posttraumatic stress disorder (PTSD).

The stress-induced affective disorder may be selected from the groupconsisting of: depressive-like behavior and associated affectivedisorders, anhedonic behavior and associated affective disorders,anxiety and associated affective disorders, cognitive impairments anddeficits and associated disorders, and combinations thereof.

The stress-induced affective disorder may comprise stress-inducedpsychopathology. The the stress-induced psychopathology may comprisedepressive and/or anxious behavior.

The subject may be a mammal, such as a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Prophylactic ketamine buffers the fear response and altersa significant number of metabolites in the brain. (A) Experimentaldesign. (B) Prophylactic ketamine does not alter CFC training behavioras measured by freezing. (C) Prophylactic ketamine administrationdecreases freezing behavior upon context re-exposure when compared withprophylactic saline administration. (n=9-10 male mice per group). Errorbars represent±SEM. * p<0.05. Sal, saline; K, ketamine; CFC, contextualfear conditioning; RE, re-exposure; HPC, hippocampus; PFC, prefrontalcortex; Sac, sacrifice; QToF, quadrupole time-of-flight; LCMS, liquidchromatography mass spectrometry.

FIGS. 2A-2E. Prophylactic ketamine significantly alters metabolites inthe PFC and HPC following stress. (A) A significant number ofmetabolites were changed in both brain regions and in both hemispheres.A total number of 8 metabolites were changed in both brain regions andin both hemispheres. (B) A pathway analysis of changed metabolites inthe PFC. Purine metabolism; phenylalanine, tyrosine, and tryptophanmetabolism; and phenylalanine metabolism are most significantly changedin the PFC following prophylactic ketamine administration. (C) A pathwayanalysis of changed metabolites in the HPC. Purine metabolism; alanine,aspartate, and glutamate metabolism; and glutamine and glutamatemetabolism are most significantly changed in the HPC followingprophylactic ketamine administration. (D) A heat map of metaboliteschanged in the PFC. (E) A heat map of metabolites changed in the HPC.(n=9-10 male mice per group). HPC, hippocampus; PFC, prefrontal cortex;ATP, adenosine triphosphate; NADH, nicotinamide adenine dinucleotide;UTP, uridine triphosphate; GDP, guanosine diphosphate; GTP, guanosinetriphosphate; AMP, adenosine monophosphate; IMP, inosine monophosphate;dUDP, deoxyuridine-diphosphate; 13-HOTE, 13-OH-9Z, 11E,15Z-octadecatrienoic acid; NAD, nicotinamide adenine dinucleotide; CDP,cytidine diphosphate; ADP, adenosine diphosphate.

FIGS. 3A-3L. Prophylactic ketamine significantly alters purinemetabolism in the PFC and HPC following stress. Purine precursors aresignificantly decreased and nucleotides are significantly increased inboth hemispheres of the (A-G) PFC and (H-L) HPC following prophylacticketamine administration. (n=9-10 male mice per group). Error barsrepresent ±SEM. * p<0.05, ** p<0.01, *** p<0.001. Sal, saline; K,ketamine. (A-G) Left/white: Sal; left/black: K (30 mg/kg). Right/gray:Sal; right/black: K (30 mg/kg). (H-L) Left/white: Sal; left/black: K (30mg/kg). Right/light gray (on the left of “Right”): Sal; right/dark gray(on the right of “Right”): K (30 mg/kg).

FIGS. 4A-4J. Prophylactic ketamine significantly alters pyrimidinemetabolism in the PFC and HPC following stress. (A-E) Pyrimidinemetabolites are significantly altered in both hemispheres of the PFCfollowing prophylactic ketamine administration. (F-H) Pyrimidinemetabolites are significantly altered in both hemispheres of the HPCfollowing prophylactic ketamine administration. (I-J) The amount of dUDPin both hemispheres of the PFC, but not the HPC, is positivelycorrelated with freezing levels upon context re-exposure in CFC in miceadministered prophylactic ketamine and stress. (n=9-10 male mice pergroup). Error bars represent±SEM. * p<0.05, ** p<0.01, *** p<0.001. Sal,saline; K, ketamine; PFC, prefrontal cortex; HPC, hippocampus; dUDP,deoxyuridine-diphosphate. (A-E) Left/white: Sal; left/black: K (30mg/kg). Right/gray: Sal; right/black: K (30 mg/kg). (F-H) Left/white:Sal; left/black: K (30 mg/kg). Right/light gray (on the left of“Right”): Sal; right/dark gray (on the right of “Right”): K (30 mg/kg).

FIGS. 5A-5Q. Prophylactic ketamine significantly alters purine andpyrimidine metabolism in plasma following stress. (A) A pathway analysisof changed metabolites in the plasma. Purine metabolism; pyrimidinemetabolism; and the TCA cycle were most significantly changed followingprophylactic ketamine administration. (B) A heat map of changedmetabolites in the plasma. (C-L) Purine metabolites significantlyincreased in the plasma following prophylactic ketamine administration.(M-Q) Pyrimidine metabolites significantly increased in the plasmafollowing prophylactic ketamine administration. (n=9-10 male mice pergroup). Error bars represent ±SEM. * p<0.05, ** p<0.01, *** p<0.001.TCA, tricarboxylic acid; Glu, glutamate; Asp, aspartate; GABA,gamma-aminobutyric acid; Ser, serine; 5-HTP, 5-hydroxytryptophan; PEP,phosphoenolpyruvic acid; TPP, thiamine pyrophosphate; AMP, adenosinemonophosphate; Ade, adenosine; MCA, monochloroacetic acid; CMP, cytidinemonophosphate; GSSG, glutathione disulfide; PE,phosphatidylethanolamine; 5′-UMP, 5′-uridine monophosphate; RLA,R-(+)-enantiomer lipoic acid; Ino, inosine; GMP, guanosinemonophosphate; 3-PGA, 3-phosphoglyceric acid; ATP, adenosinetriphosphate; NANA, N-acetylneuraminic acid; SAMe, S-adenosylmethionine; GDP, guanosine diphosphate; PRPP, 5-phospho-alpha-D-ribosyl1-pyrophosphate; TRA, tiaramide; 5′-UDP, 5′-uridine diphosphate; CYSSG,cysteine-glutatione disulfide.

FIGS. 6A-6Z, 6AA-6HH. Positive mode metabolites changed in the PFCfollowing prophylactic ketamine administration and CFC stress. (n=9-10male mice per group). Error bars represent ±SEM. * p<0.05, ** p<0.01,*** p<0.001. Left/white: Saline; left/black: Ketamine. Right/gray:Saline; right/black: Ketamine.

FIGS. 7A-7K. Negative mode metabolites changed in the PFC followingprophylactic ketamine administration and CFC stress. (n=9-10 male miceper group). Error bars represent ±SEM. * p<0.05, ** p<0.01, *** p<0.001.Left/white: Saline; left/black: Ketamine. Right/gray: Saline;right/black: Ketamine.

FIGS. 8A-8V. Positive and negative mode metabolites changed in the HPCfollowing prophylactic ketamine administration and CFC stress. (n=9-10male mice per group). Error bars represent ±SEM. * p<0.05, ** p<0.01,*** p<0.001. Left/white: Saline; left/black: Ketamine. Right/light gray(on the left of “Right”): Saline; right/dark gray (on the right of“Right”): Ketamine.

FIGS. 9A-9W. Prophylactic ketamine does not impact freezing behavior,and does not significantly alter purine or pyrimidine metabolism in thePFC and HPC. (A) Experimental design. (B-C) Prophylactic ketamine doesnot alter context exposure or context re-exposure as measured byfreezing when compared with prophylactic saline administration.

Only 3 purine metabolites are significantly altered by ketamineadministration in one hemisphere of the (D-J) PFC or (K-O) HPC. Only 2pyrimidines are significantly altered by ketamine administration in onehemisphere of the (P-T) PFC, or (U-W) HPC. (n=8-9 male mice per group).Error bars represent ±SEM. * p<0.05, ** p<0.01. Sal, saline; K,ketamine; RE, re-exposure; HPC, hippocampus; PFC, prefrontal cortex;Sac, sacrifice; QToF, quadrupole time-of-flight; LCMS, liquidchromatography mass spectrometry; min, minutes; dUDP,deoxyuridine-diphosphate. (D-J), (P-T): Left/white: Saline; left/black:Ketamine (30 mg/kg). Right/gray: Saline; right/black: Ketamine (30mg/kg). (K-O), (U-W): Left/white: Saline; left/black: Ketamine (30mg/kg). Right/light gray (on the left of “Right”): Saline; right/darkgray (on the right of “Right”): Ketamine (30 mg/kg).

FIGS. 10A-10O. Prophylactic ketamine without CFC stress does notsignificantly alter purine or pyrimidine metabolism in plasma. (A-J)Purine metabolites were not significantly changed following prophylacticketamine administration. (K-O) Pyrimidine metabolites were notsignificantly changed following prophylactic ketamine administration.(n=8-9 male mice per group). Error bars represent ±SEM.

DETAILED DESCRIPTION OF THE INVENTION

Certain individuals are particularly vulnerable to developingstress-induced psychiatric disorders. Prophylactic treatments may beable to prevent the development of such psychiatric disorders, but thereis currently no method to predict which patients are likely to benefitfrom such interventions. The present method uses therapeutic targets andbiomarkers for the development of prophylactic treatments againststress-induced psychiatric disorders. The biomarkers can be used todevelop targeted prophylactic treatments as well as blood tests topredict patient responsiveness to treatments (including pharmacologicaltreatments) for informed treatment decisions.

Up to 30% of depression patients do not respond to pharmacologicaltreatment. Khalid S A, Treatment-resistant depression: therapeutictrends, challenges, and future directions, Patient Preference andAdherence, 2012: 369-388. The present biomarkers may be used for a bloodtest to identify individuals who would be responsive to a prophylactictreatment against a stress-induced affective disorder or stress-inducedpsychopathology.

The present biomarkers may be used in drug discovery, e.g., identifyingnew targets for prophylactic treatments against stress-inducedpsychiatric disorders. This technology can enable the development ofmore targeted and effective therapies for stress-induced psychiatricdisorders.

The biomarkers may help identify individuals at risk of developingstress-related psychiatric disorders. The biomarkers may be used for adiagnostic test to discriminate between different etiologies of apsychiatric disease.

The present disclosure provides for a method for treating a subject fora stress-induced affective disorder or stress-induced psychopathology.The method may comprise the following steps: (a) administering or havingadministered a prophylactic treatment to the subject prior to astressor; (b) determining the level of one or more biomarkers in abiological sample obtained from the subject after step (a) and after thestressor; and (c) comparing the level obtained in step (b) with thelevel of the one or more biomarkers in a control sample.

The present disclosure also provides for a method for assessing aprophylactic treatment of a subject for a stress-induced affectivedisorder or stress-induced psychopathology. The method may comprise thefollowing steps: (a) determining the level of one or more biomarkers ina biological sample obtained from the subject after a prophylactictreatment and after a stressor; and (b) comparing the level obtained instep (a) with the level of the one or more biomarkers in a controlsample.

The method may further comprise maintaining a treatment regime ordetermining the prophylactic treatment as being effective, when thelevel of one or more biomarkers increases or decreases by at least 10%compared to its level in the control sample.

An additional step of the present method may alternatively be adjustinga treatment regime, when the level of one or more biomarkers isunchanged, or increases or decreases by less than 10%, compared to itslevel in the control sample.

In certain embodiments, the present method determines/detects the levelof one or more biomarkers selected from those listed in FIGS. 2B-2E,3A-3L, 4A-4J, 5A-5Q, 6A-6Z, 6AA-6HH, 7A-7K, 8A-8V, 9A-9W, 10A-10O, andTables 1-3, and combinations thereof. In certain embodiments, thepresent method determines/detects the level of one or more biomarkersselected from those listed in any of FIGS. 1A-1C, 2A-2E, 3A-3L, 4A-4J,5A-5Q, 6A-6Z, 6AA-6HH, 7A-7K, 8A-8V, 9A-9W, 10A-10O, and Tables 1-3, andcombinations thereof.

If the level of at least one, or at least 2 (at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 20, at least 30, at least 40, at least 50, between 5 and 30,between 5 and 10, between 10 and 20, between 30 and 50, or between 50and 100) biomarkers increases or decreases by about 1% to about 100%,about 5% to about 90%, about 10% to about 80%, about 5% to about 70%,about 5% to about 60%, about 10% to about 50%, about 15% to about 40%,about 5% to about 20%, about 1% to about 20%, about 10% to about 30%,about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 100%, about 10% to about90%, about 12.5% to about 80%, about 20% to about 70%, about 25% toabout 60%, or about 25% to about 50%, about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about9-fold, about 10-fold, at least 1.1-fold, at least 1.2-fold, at least1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, atleast 1.8-fold, at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 120-fold, from about 2-foldto about 500-fold, from about 1.1-fold to about 10-fold, from about1.1-fold to about 5-fold, from about 1.5-fold to about 5-fold, fromabout 2-fold to about 5-fold, from about 3-fold to about 4-fold, fromabout 5-fold to about 10-fold, from about 5-fold to about 200-fold, fromabout 10-fold to about 150-fold, from about 10-fold to about 20-fold,from about 20-fold to about 150-fold, from about 20-fold to about50-fold, from about 30-fold to about 150-fold, from about 50-fold toabout 100-fold, from about 70-fold to about-150 fold, from about100-fold to about 150-fold, from about 10-fold to about 100-fold, fromabout 100-fold to about 200-fold, compared to its (or their) level in acontrol sample, the therapy is considered to be effective. An effectivetherapy may be maintained/continued, or discontinued if the patient'scondition has improved and is no longer in need of treatment. Anineffective treatment may be altered or modified, or replaced with othertreatment.

The present methods can include the steps of measuring the level of atleast one biomarker in a sample from a patient having received, orreceiving, a therapeutic intervention, and comparing the measured levelto a reference level or the level of at least one biomarker in a controlsample. The measured level of the at least one biomarker is indicativeof the therapeutic efficacy of the therapeutic intervention.

Based on the measured biomarker levels, therapy may be continued oraltered, e.g., by change of dose or dosing frequency, or by addition ofother active agents, or change of therapeutic regimen altogether.

The present invention also encompasses a method of predicting orassessing the level of severity of the disorder/condition in a patient.In one embodiment, the method comprises measuring the level of at leastone biomarker in a biological sample from a patient; and comparing themeasured level to a reference level or the level of the at least onebiomarker in a control sample, wherein the measured level of the atleast one biomarker is indicative of the level of severity of thedisorder/condition in the patient. In other embodiments, an increase ordecrease (as described herein) in the level of the biomarker isindicative of the level of severity of the disorder/condition in thepatient.

The level/amount of the biomarker(s) in a patient may bedetermined/detected. The level/amount of the biomarker(s) of the patientmay be compared with a reference value, where the reference value isbased on the level/amount of a set of biomarkers in a control sample,and/or based on a set of biomarkers in an unaffected individual orunaffected individuals, and/or based on a set of biomarkers in thepatient before, after and/or during therapy. The changes in biomarkerlevel may be used to alter or direct therapy, including, but not limitedto, initiating, altering or stopping therapy.

Another aspect of the disclosure is a kit containing a reagent formeasuring at least one biomarker in a biological sample, instructionsfor measuring at least one biomarker, and instructions for evaluating ormonitoring therapeutic efficacy in a patient based on the level of theat least one biomarker. In some embodiments, the kit contains reagentsfor measuring from 1 to about 20 biomarkers, including 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 up to n biomarkers.Also encompassed by the disclosure are kits for assessing or predictingthe severity or progression of the disorder/condition in a subject. Thekit may comprise a reagent for measuring at least one biomarker in abiological sample, and instructions for assessing severity orprogression of the disorder/condition based on the level of the at leastone biomarker. The kit may comprise one or biochips to assay the levelsof a plurality biomarkers.

The level of at least one, or at least 2 (or at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 20, at least 30, at least 40, between 5 and 30, between 5 and 10,between 2 and 6, between 3 and 5, between 10 and 20, or between 20 and45) biomarkers in the sample may increase or decrease by about 1% toabout 100%, about 5% to about 90%, about 10% to about 80%, about 5% toabout 70%, about 5% to about 60%, about 10% to about 50%, about 15% toabout 40%, about 5% to about 20%, about 1% to about 20%, about 10% toabout 30%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 100%, about 10% toabout 90%, about 12.5% to about 80%, about 20% to about 70%, about 25%to about 60%, or about 25% to about 50%, about 2-fold, about 3-fold,about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold,about 9-fold, about 10-fold, at least 1.1-fold, at least 1.2-fold, atleast 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold,at least 1.8-fold, at least 2-fold, at least 2.5-fold, at least 3-fold,at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold,at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, atleast 50-fold, at least 100-fold, at least 120-fold, from about 2-foldto about 500-fold, from about 1.1-fold to about 10-fold, from about1.1-fold to about 5-fold, from about 1.5-fold to about 5-fold, fromabout 2-fold to about 5-fold, from about 3-fold to about 4-fold, fromabout 5-fold to about 10-fold, from about 5-fold to about 200-fold, fromabout 10-fold to about 150-fold, from about 10-fold to about 20-fold,from about 20-fold to about 150-fold, from about 20-fold to about50-fold, from about 30-fold to about 150-fold, from about 50-fold toabout 100-fold, from about 70-fold to about 150-fold, from about100-fold to about 150-fold, from about 10-fold to about 100-fold, fromabout 100-fold to about 200-fold, compared to the level(s) in thecontrol sample.

In certain embodiments, the levels of a plurality of biomarkers in thesample may be assayed, which comprises 2 or more, 3 or more, 4 or more,5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 3-50, 5-50, 10-50,15-50, 20-50, 30-50, or 50-100, biomarkers.

The samples may include, but are not limited to, serum, plasma, blood,whole blood and derivatives thereof, cardiac tissue, bone marrow, urine,cerebrospinal fluid (CSF), myocardium, endothelium, skin, hair, hairfollicles, saliva, oral mucus, vaginal mucus, sweat, tears, epithelialtissues, semen, seminal plasma, prostatic fluid, excreta, ascites,lymph, bile, as well as other samples or biopsies. In one embodiment,the biological sample is plasma or serum.

The level or amount of a biomarker in a patient sample can be comparedto a reference level or amount of the biomarker present in a controlsample. The control sample may be from a patient who has experienced astressor without a prophylactic treatment, or from a plurality ofpatients who have experienced a stressor without a prophylactictreatment.

The control sample may be from a patient who has had a prophylactictreatment without experiencing a stressor, or a plurality of patientswho have had a prophylactic treatment without experiencing a stressor.The control sample may be from a healthy subject or a plurality ofhealthy subjects. In other embodiments, a control sample is taken from apatient prior to a stressor or prior to a prophylactic treatment (froman untreated patient). In certain embodiments, a control sample is froma patient or a plurality of patients unresponsive to a prophylactictreatment. Reference levels for a biomarker can be determined bydetermining the level of a biomarker in a sufficiently large number ofsamples obtained from normal, healthy control subjects to obtain apre-determined reference or threshold value. A reference level can alsobe determined by determining the level of the biomarker in a sample froma patient prior to a stressor or prior to a prophylactic treatment (froman untreated patient). Reference (or calibrator) level information andmethods for determining reference levels can be obtained from publiclyavailable databases, as well as other sources.

The present methods may comprise prophylactically treating astress-induced affective disorder or stress-induced psychopathology in asubject. Also encompassed by the present methods are inducing and/orenhancing stress resilience in a subject. In certain embodiments, aneffective amount of an antagonist of the glutamate N-methyl-D-aspartate(NMDA) receptor, such as ketamine or a pharmaceutically acceptable saltor derivative thereof, is administered to a subject prior to a stressor.

The present agent/composition may be administered therapeutically toachieve a therapeutic benefit or prophylactically to achieve aprophylactic benefit. By therapeutic benefit is meant eradication oramelioration of the underlying stress-induced affective disorder beingtreated, and/or eradication or amelioration of one or more of thesymptoms associated with the underlying disorder. By prophylacticbenefit is meant prevention or delay of the onset of a stress-inducedaffective disorder, and/or prevention or delay of the onset of one ormore of the symptoms associated with a stress-induced affectivedisorder. In certain embodiments, an effective amount of the presentagent/composition to be administered prevents stress-related disordersfrom developing or being exacerbated into more serious conditions.

In certain embodiments, for prophylactic administration, the presentagent/composition may be administered to a patient at risk of developinga stress-induced affective disorder, or to a patient reporting one ormore of the physiological symptoms of a stress-induced affectivedisorder, even though a diagnosis of a stress-induced affective disordermay not have yet been made. In certain embodiments, prophylacticadministration is applied to avoid the onset of the physiologicalsymptoms of the underlying disorder, before the symptom manifestscyclically. In this latter embodiment, the therapy is prophylactic withrespect to the associated physiological symptoms instead of theunderlying indication. In certain embodiments, the presentagent/composition is administered prior to recurrence of a stressor. Incertain embodiments, the present agent/composition is administered priorto the onset of a particular symptom.

In a further embodiment, the present invention provides for the use ofthe present agent or a pharmaceutically acceptable salt or solvatethereof, or a physiologically functional derivative thereof, in thepreparation of a medicament for the treatment of a stress-inducedaffective disorder.

“Treating” or “treatment” of a state, disorder or condition includes:

(1) preventing or delaying the appearance of clinical symptoms of thestate, disorder, or condition developing in a person who may beafflicted with or predisposed to the state, disorder or condition butdoes not yet experience or display clinical symptoms of the state,disorder or condition; or(2) inhibiting the state, disorder or condition, i.e., arresting,reducing or delaying the development of the disease or a relapse thereof(in case of maintenance treatment) or at least one clinical symptom,sign, or test, thereof; or(3) relieving the disease, i.e., causing regression of the state,disorder or condition or at least one of its clinical or sub-clinicalsymptoms or signs.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. In certain embodiments, since a prophylactic doseis used in subjects prior to or at an earlier stage of a disorder, theprophylactically effective amount is less than the therapeuticallyeffective amount. In certain embodiments, the prophylactically effectiveamount is similar to, identical to, or more than, the therapeuticallyeffective amount.

A therapeutically effective amount, or an effective amount, of a drug isan amount effective to demonstrate a desired activity of the drug. A“therapeutically effective amount” will vary depending on the compound,the disorder and its severity and the age, weight, physical conditionand responsiveness of the subject to be treated. In certain embodiments,a effective amount of ketamine, or a pharmaceutically acceptable salt orsolvate thereof, or a physiologically functional derivative thereof, isan amount effective to prevent or delay the onset of a stress-inducedaffective disorder, and/or effective to alleviate, one or more of thesymptoms of a stress-induced affective disorder.

In certain embodiments, an effective amount of the present agent is asub-anesthetic amount of ketamine, or a pharmaceutically acceptable saltor solvate thereof, or a physiologically functional derivative thereof.In certain embodiments, an effective amount of the present agent is asub-analgesic amount of ketamine, or a pharmaceutically acceptable saltor solvate thereof, or a physiologically functional derivative thereof.

A subject may be treated with ketamine, or a pharmaceutically acceptablesalt or solvate thereof, or a physiologically functional derivativethereof, via intravenous, oral, transdermal or intranasaladministration. In certain embodiments, a subject is injected withketamine, or a pharmaceutically acceptable salt or solvate thereof, or aphysiologically functional derivative thereof.

A subject may be treated with a single dose of an effective amount ofketamine, or a pharmaceutically acceptable salt or solvate thereof, or aphysiologically functional derivative thereof, prior to and/or after astressor. In some aspects, a subject is treated with multiple doses ofan effective amount of ketamine, or a pharmaceutically acceptable saltor solvate thereof, or a physiologically functional derivative thereof,prior to, during, and/or after, a stressor.

In certain embodiments, the present agent, such as ketamine, or apharmaceutically acceptable salt or solvate thereof, or aphysiologically functional derivative thereof, is administered in acomposition comprising a pharmaceutically acceptable carrier, excipientor diluent. Also provided herein is a pharmaceutical composition thatcomprises ketamine, or a pharmaceutically acceptable salt or solvatethereof, or a physiologically functional derivative thereof, and apharmaceutically acceptable carrier, excipient or diluent, for use inthe prophylactic treatment of a stress-induced affective disorder.

“Patient” or “subject” refers to mammals and includes human andveterinary subjects. In certain embodiments, the subject is a mammal.

Biomarkers

The present method measures the level of at least one biomarker in abiological sample.

The biomarkers may be purines, purine metabolites, purine precursors,purine derivatives, pyrimidines, pyrimidine metabolites, pyrimidineprecursors, pyrimidine derivatives, nucleotides, nucleotide metabolites,nucleotide precursors, nucleotide derivatives, neurotransmitters,neurotransmitter metabolites, neurotransmitter precursors, andneurotransmitter derivatives. Neurotransmitters may be inhibitoryneurotransmitters or excitatory neurotransmitters. Neurotransmitters maybe amino acid-derived neurotransmitters. Neurotransmitter precursors maybe precursors to inhibitory neurotransmitters or precursors toexcitatory neurotransmitters.

In one embodiment, the biomarkers include purine metabolites. Thebiomarkers may be selected from those listed in FIGS. 5C-5L. In oneembodiment, the biomarkers include pyrimidine metabolites. Thebiomarkers may be selected from those listed in FIGS. 5M-5Q.

Biomarkers may include purine metabolites, such as adenine, adenosinemonophosphate (AMP), adenosine triphosphate (ATP), D-ribose 5-phosphate,D-ribulose 5-phosphate, guanosine monophosphate, guanosine diphosphate,hypoxanthine, inosine, and phosphoribosyl pyrophosphate.

The level of the biomarker in the biological sample may increase ordecrease compared to the level of the biomarker in the control sample.

Biomarkers may include pyrimidine metabolites, such as cytidinemonophosphate, dihydrothymine, uridine 5-monophosphate, and uridine5-diphosphate. Biomarkers may include pyrimidine metabolites, such as5,6-dihydrouridine.

In one embodiment, the level of the biomarker in the biological sampleincreases compared to the level of the biomarker in the control sample.Non-limiting examples of such biomarkers include adenine, adenosinemonophosphate (AMP), adenosine triphosphate (ATP), D-ribose 5-phosphate,D-ribulose 5-phosphate, guanosine monophosphate, guanosine diphosphate,hypoxanthine, inosine, and phosphoribosyl pyrophosphate. Non-limitingexamples of such biomarkers also include cytidine monophosphate,dihydrothymine, uridine 5-monophosphate, and uridine 5-diphosphate.

In one embodiment, the level of the biomarker in the biological sampledecreases compared to the level of the biomarker in the control sample.Non-limiting examples of such biomarkers include 5,6-dihydrouridine.

In one embodiment, the biomarkers include amino acid-derivedneurotransmitters and precursors. The biomarkers may be selected fromthose listed in Table 3.

Biomarkers may include excitatory neurotransmitters such as serine(e.g., D-serine), glutamic acid (e.g., L-glutamic acid), and glutamate.Biomarkers may include inhibitory neurotransmitters such asgamma-aminobuytric acid (GABA), 5-hydroxy-L-tryptophan (5-HTP),O-phosphoethanolamine (PE), and N-acetyl-L-tyrosine.

In one embodiment, the level of the biomarker in the biological sampleincreases compared to the level of the biomarker in the control sample.Non-limiting examples of such biomarkers include 5-hydroxy-L-tryptophan(5-HTP), and O-phosphoethanolamine (PE).

In another embodiment, the level of the biomarker in the biologicalsample decreases compared to the level of the biomarker in the controlsample. Non-limiting examples of such biomarkers include serine (e.g.,D-Serine), glutamic acid (e.g., L-glutamic acid), glutamate,gamma-aminobuytric acid (GABA), and N-acetyl-L-tyrosine.

In certain embodiments, the sample is a body fluid. For example, thebody fluid can include, but are not limited to, serum, plasma, blood,whole blood and derivatives thereof, urine, tears, saliva, sweat,cerebrospinal fluid (CSF), oral mucus, vaginal mucus, seminal plasma,semen, prostatic fluid, excreta, ascites, lymph, bile, and amnioticfluid. In certain embodiments, the biological sample is plasma or serum.In certain embodiment, samples can include, but are not limited to,skin, hair, hair follicles, epithelial tissues, bone marrow,endothelium, brain tissue, as well as other samples or biopsies.

The sample may be obtained at any time point after, during, and/orbefore, the prophylactic treatment and/or after a stressor, such asabout 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 10 hours, about 12 hours, about 15 hours, about 18 hours,about 20 hours, about 22 hours, about 1 day, about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks,about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3months, about 4 months, about 5 months, about 6 months, about 1 year,about 2 years, about 3 years, about 5 years or longer after, during,and/or before, the prophylactic treatment and/or after the stressor. Thetime point may also be earlier or later. In one embodiment, the sampleis obtained about 4 weeks after the prophylactic treatment and/or afterthe stressor.

In certain embodiments, 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 orall, biomarkers selected from the biomarkers in FIGS. 2B-2E, 3A-3L,4A-4J, 5A-5Q, 6A-6Z, 6AA-6HH, 7A-7K, 8A-8V, 9A-9W, 10A-10O, and Tables1-3, and combinations thereof, are measured. In certain embodiments, 1or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 ormore, 30 or more, 35 or more, 40 or more, 45 or all, biomarkers selectedfrom the biomarkers in FIGS. 1A-1C, 2A-2E, 3A-3L, 4A-4J, 5A-5Q, 6A-6Z,6AA-6HH, 7A-7K, 8A-8V, 9A-9W, 10A-10O, and Tables 1-3, and combinationsthereof, are measured. In some embodiments, a panel of no greater than20, no greater than 15, no greater than 10, or no greater than 5biomarkers is tested, the panel including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the biomarkers asdescribed herein.

The level or amount of biomarkers in a patient sample can be compared toa reference level or amount of the biomarkers present in a controlsample.

The level of a biomarker (or a plurality of biomarkers) can be detectedand/or quantified by any of a number of methods well known to those ofskill in the art. The biomarkers may be detected by, for example, massspectrometry (MS). Also included are analytic biochemical methods suchas electrophoresis, capillary electrophoresis, liquid chromatography,nuclear magnetic resonance (NMR), spectrophotometry, high-performanceliquid chromatography (HPLC), thin layer chromatography (TLC),hyper-diffusion chromatography, liquid chromatography-tandem massspectrometry, electrochemical analysis, and the like. Other techniquesinclude refractive index spectroscopy (RI), ultra-violet spectroscopy(UV), fluorescent analysis, radiochemical analysis, near-infraredspectroscopy (Near-IR), light scattering analysis (LS),gas-chromatography-mass spectroscopy (GC-MS), gas chromatography, massspectrometry, and liquid-chromatography-mass spectroscopy (LC-MS,LC-MS/MS) and other methods known in the art, alone or in combination.U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168. Methodsin Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed.Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7thEdition, Stites & Terr, eds. (1991).

Levels of biomarkers can be detected using methods well known in the artas a reflection of metabolic activity, such as liquid chromatography.Liquid chromatography coupled with tandem mass spectrometric detection(LC/MS/MS) can be used as an analytical method. Using automatedsample-processing techniques, such as on-line column switching, combinedwith high-sample-density microtiter plates, can further maximizeanalytical throughput. Selectivity can be further enhanced by thequadrupole ion trap, a device that “traps” ions in a space bounded by aseries of electrodes. The unique feature of the ion trap is that anMS/MS experiment (or, multi-step MS experiments) can be performedsequentially in time within a single mass analyzer, yielding a wealth ofstructural information. Hybrid quadrupole-time-of-flight (Q-TOF)LC/MS/MS systems can also be used for the characterization of metaboliteprofiles. The configuration of Q-TOF results in high sensitivity in massresolution and mass accuracy in a variety of scan modes.

Liquid chromatography coupled with nuclear magnetic resonancespectroscopy (LC-NMR) provides a way of confirming absolute molecularconfigurations. A linear ion-trap mass spectrometer possessessignificantly enhanced production-scanning capabilities, while retainingall of the scan functions of a triple quadrupole MS. The ultra-highresolution and sensitivity of Fourier transform ion-cyclotron resonanceMS (FI-ICRMS) can be useful for the analysis and characterization ofbiological mixtures.

HPLC columns equipped with coulometric array technology can be used toanalyze the samples.

The compounds may be mapped to biochemical pathways and the pathwaysthat are affected are determined.

The present method may comprise studying metabolites in the sample,conducting metabolomics analysis (including targeted or untargetedmetabolomic profiling), and/or analyzing metabolite profiles of thesample.

The disclosure further includes arrays, microarrays, chips, biochipsetc. for the analysis of levels of a plurality of biomarkers.

In one embodiment, a difference (increase or decrease) in the measuredlevel of the biomarker(s) relative to the level of the biomarker(s) inthe control sample or a pre-determined reference value is indicative ofthe therapeutic efficacy of the therapeutic intervention (e.g., aprophylactic treatment). In another embodiment, an increase (ordecrease) in the measured level of the biomarker(s) relative to thelevel of the biomarker(s) in the control sample or pre-determinedreference value is indicative of the therapeutic efficacy of thetherapeutic intervention. For instance, in such embodiments, when thelevel of one or more biomarkers is increased (or decreased) whencompared to the level in a control sample or pre-determined referencevalue in response to a therapeutic intervention, the increase (ordecrease) is indicative of therapeutic efficacy of the therapeuticintervention.

In certain embodiments, a reduction or decrease in the measured level ofthe biomarker(s) relative to the level of the biomarker(s) in thecontrol sample or pre-determined reference value can be indicative ofthe therapeutic efficacy of the therapeutic intervention.

For instance, in such embodiments, when the level of one or morebiomarkers is decreased (or increased) when compared to the level in acontrol sample or pre-determined reference value in response to atherapeutic intervention, the decrease (or increase) is indicative oftherapeutic efficacy of the therapeutic intervention.

Patients showing different (elevated or reduced) levels of one or morebiomarkers can be identified. The expression profile of these biomarkersmay be used to calculate a score for the combined or individualbiomarker expression. The scores of these patients will be compared tothe score of individuals as a control. The clinical condition of thesepatients with respect to their psychiatric status may be correlated withthe biomarker profiles. The scores may be used to identify groups ofpatients responsive to the prophylactic treatment.

Samples Sampling methods are well known by those skilled in the art andany applicable techniques for obtaining biological samples of any typeare contemplated and can be employed with the methods of the presentinvention. (See, e.g., Clinical Proteomics: Methods and Protocols, Vol.428 in Methods in Molecular Biology, Ed. Antonia Vlahou (2008).) Thesamples may be drawn before, during or after a prophylactic treatmentand/or a stressor. The samples may be drawn at different time pointsbefore, during or after a prophylactic treatment and/or a stressor.

The sample may be obtained from the subject at any point before, duringor after a prophylactic treatment and/or a stressor. In someembodiments, the sample is obtained about 1 week, about 2 weeks, about 3weeks, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, about 6 months, at least 1, 2, 3, or 6 months, before,during or after a prophylactic treatment and/or a stressor. In someembodiments, the sample is obtained least 1, 2, 3, 4, 6 or 8 weeksbefore, during or after a prophylactic treatment and/or a stressor. Insome embodiments, the sample is obtained at least 1, 2, 3, 4, 5, 6, or 7days before, during or after a prophylactic treatment and/or a stressor.In some embodiments, the sample is obtained at least 10 minutes, 30minutes, 1 hour, 6 hours, 12 hours, 18 hours or 24 hours before, duringor after a prophylactic treatment and/or a stressor. In otherembodiments, the sample is obtained at least one week before, during orafter a prophylactic treatment and/or a stressor. In some embodiments,one or more biomarkers are measured between 1 and 8 weeks, between 2 and7 weeks, at 1, 2, 3, 4, 5, 6, 7 or 8 weeks before, during or after aprophylactic treatment and/or a stressor.

Ketamine

Ketamine ((RS)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone) is anantagonist of the glutamate N-methyl-D-aspartate (NMDA) receptor(NMDAR). Ketamine also acts on opioid receptors, sigma receptors,muscarinic receptors, monoamine transporters, etc.

Ketamine is a chiral compound. As used herein, the term “ketamine” mayrefer to (S)-ketamine (also referred to as S(+)-ketamine or esketamine),(R)-ketamine (R(−)-ketamine), or a racemic mixture of (S)-ketamine and(R)-ketamine. In certain embodiments, the ketamine compositions containdifferent proportions of the S(+) and R(−) stereoisomers. In certainembodiments, the ketamine compositions contain only (S)-ketamine or(R)-ketamine, or are enantiomerically enriched for a ketamineenantiomer. In certain embodiments, the ketamine composition is enrichedto contain, for example, greater than 60%, greater than 70%, greaterthan 80%, greater than 90%, greater than 95%, greater than 99%, orgreater than 99.9 of (S)-ketamine or (R)-ketamine. Paul et al.,“Comparison of racemic ketamine and S-ketamine in treatment-resistantmajor depression: report of two cases”, World J. of Bio. Psych., 2009,pp 241-244, Vol. 10(3); Paskalis et al., Oral Administration of the NMDAReceptor Antagonist S-Ketamine as Add-on Therapy of Depression: A CaseSeries, Pharmacopsychiatry, 2010, pp 33-35, Vol. 40; Noppers et al.,Absence of long-term analgesic effect from a short-term S-ketamineinfusion on fibromyalgia pain: A randomized, prospective, double blind,active placebo-controlled trial”, Eur. J. of Pain., 2011, 15(9):942-9;Matthews et al., Ketamine for Treatment-Resistant Unipolar Depression,CNS Drugs, 2012, 1-16; and International Patent Publication No.WO2013138322.

The term “pharmaceutically acceptable derivative” refers to anypharmaceutically acceptable salt, solvate or prodrug, e.g. ester, of acompound which upon administration to the recipient is capable ofproviding (directly or indirectly) a compound or an active metabolite orresidue thereof. Such derivatives are recognizable to those skilled inthe art, without undue experimentation. Derivatives are described, forexample, in Burger's Medicinal Chemistry and Drug Discovery, 5thEdition, Vol 1: Principles and Practice, which is incorporated herein byreference. In certain embodiments, pharmaceutically acceptablederivatives include salts, solvates, esters, carbamates, and phosphateesters. In one embodiment, the present composition contains ahydrochloride salt of ketamine.

The present agent may be administered by various routes, includingintravenous (i.v. or IV), intranasal (i.n. or IN), intramuscular (i.m.or IM), caudal, intrathecal, and subcutaneous (s.c.) routes.

NMDA Receptor Antagonists—Ketamine and Other Compounds

NMDA receptor antagonists are compounds that antagonize, or inhibit, theaction of the NMDA receptor. An NMDA receptor antagonist may be acompetitive antagonist, an uncompetitive antagonist, a noncompetitiveantagonist, and/or a glycine antagonist.

Non-limiting examples of NMDA receptor antagonists include, ketamine,dextromethorphan (DXM), histogranin, memantine, meperidine, methadone,methoxetamine (MXE), phencyclidine (PCP), nitrous oxide (N₂O), AP5 (APV,R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene ((3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), Selfotel, Amantadine, Atomoxetine, AZD6765, Agmatine, chloroform,dextrallorphan, dextromethorphan, dextrorphan, diphenidine, dizocilpine(MK-801), ethanol, eticyclidine, gacyclidine, ibogaine, magnesium,memantine, nitromemantine, rolicyclidine, tenocyclidine, methoxydine,tiletamine, neramexane, eliprodil, dexoxadrol, etoxadrol, remacemide,delucemine, WMS-2539, NEFA, 8A-PDHQ, HU-211, Aptiganel (Cerestat,CNS-1102), rhynchophylline, kynurenic acid, Rapastinel (GLYX-13),NRX-1074, 7-Chlorokynurenic acid, 4-Chlorokynurenine (AV-101), TK-40,1-Aminocyclopropanecarboxylic acid (ACPC), L-Phenylalanine, Xenon, oranalogs or derivatives thereof. Ketamine derivatives such as Rapastinelor Glyx-13 are also included. Rapastinel is an NMDA receptor glycinesite partial agonist. It is an amidated tetrapeptide(Thr-Pro-Pro-Thr-NH₂) which rapidly crosses the blood brain barrier, butis not active orally.

Compounds that are mechanistically similar to ketamine are expected tobe protective against stress-induced de novo psychopathology. Suchcompounds include:

-   -   Ro 25-6981, a GluN2B-selective antagonist (Miller Ohio, et al.        (2014), eLife 3:e03581), which has been shown to have rapid        antidepressant actions in rodent models of depression.    -   CP-101,606, a GluN2B-selective antagonist (Preskorn S, et al.        (2007): A placebo-controlled trial of the NR2B specific NMDA        antagonist CP-101, 606 plus paroxetine for treatment resistant        depression (TRD). American Psychological Association meeting),        which has been shown to be protective in animal models of brain        injury and stroke.    -   GLYX-13, a novel N-methyl-D-aspartate receptor (NMDAR)        glycine-site functional partial agonist and rapid-acting        antidepressant (Burgdorf J, et al. (2013),        Neuropsychopharmacology 38:729-42). GLYX-13 received        Breakthrough Therapy designation from the U.S. Food and Drug        Administration (FDA) for adjunctive treatment of MDD in January,        2016, and    -   CX546 (Tocris), an ampakine (an AMPA receptor agonist) (Zhou W,        et al. (2014), Eur. Psychiatry 29:419-23), which relieves the        respiratory depression induced by fentanyl.

Non-limiting examples of the NMDA receptor antagonists also includeanti-receptor antibodies, anti-ligand antibodies, etc.

Several synthetic opioids function as NMDA receptor-antagonists, such aspethidine, methadone, meperidine, dextropropoxyphene, tramadol,levorphanol, and ketobemidone.

AMPA Receptor Agonists

AMPA receptor agonists are compounds that activate the action of thea-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Itis expected that compounds that activate the AMPA receptor, includingmetabolites, will have a similar effect as the present effects shownwith ketamine, in view of recent findings that a ketamine metabolite'santidepressant activity in mice was due to sustained activation of theAMPA receptor, rather than inhibiting NMDAR. (See, Zanos et al., (2016).“NMDAR inhibition-independent antidepressant actions of ketaminemetabolites. Nature, 533: 481-486.)

Thus, in certain embodiments, AMPA receptor agonists may be used in themethods described herein. Non-limiting examples of the AMPA receptoragonists include glutamate, AMPA, 5-fluorowillardiine, domoic acid,quisqualic acid, (2R,6R)-hydroxynorketamine, CX546, etc.

Ketamine Metabolites

Ketamine is a derivative of arylcyclohexylamine and contains a chiralcenter. Since the 1950s, a large number of arylcyclohexylamines havebeen synthesized: these compounds have shown a wide range of possiblepharmacological activities. When administered orally, it undergoesfirst-pass metabolism, where it is stereo selectively metabolized into abroad array of metabolites, including norketamine, hydroxyketamines,dehydronorketamine and hydroxynorketamine (HNK). After ketamineadministration, (2S,6S;2R,6R)-HNK are the two major HNK metabolitesfound in the plasma and brain. Interestingly, a recent study has shownthat the (2R,6R)-HNK metabolite is: 1) essential for the antidepressanteffects of ketamine, 2) dependent ona-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptoractivation, and 3) non-hypnotic (Zanos et al., 2016). All of thesecompounds are expected to behave similarly in the presently describedmethods, including enantiomers and non-psychotomimetic metabolites ofketamine.

The present composition may also comprise ketamine's enantiomers and/ornon-psychotomimetic metabolites. Such compounds include:

-   -   1. (2R,6R)-HNK, a metabolite of ketamine that may mediate the        antidepressant effects of ketamine and lacks the        ketamine-related side effects (Zanos et al., 2016)    -   2. (2S,6S)-HNK, a metabolite of ketamine (Zanos et al., 2016,        synthesis of these compounds (2R,6R)-HNK and (2S,6S)-HNK are        described in Zanos et al. 2016 and Wainer et al. WO 2013/056229        (2013), The use of (2R,6R)-hydroxynorketamine,        (S)-dehydronorketamine and other stereoisomeric dehydro and        hydroxylated metabolites of (R,S)-ketamine in the treatment of        depression and neuropathic pain).    -   3. (R)-ketamine, the R-enantiomer of ketamine, which has        rapid-onset and sustained antidepressant effects without        psychotomimetic side effects (Yang et al., 2015), and    -   4. (S)-ketamine, the S-enantiomer of ketamine, which is being        developed as an intranasal spray, currently in phase III        clinical trials for treatment-resistant depression.

Other ketamine analogs may also be used. Such compounds include:

-   -   5. Fluorodeschloroketamine, an analog of ketamine where the        chlorine (Cl) group has been replaced by fluorine (F), and    -   6. Tiletamine, an analog of ketamine commonly used as a        veterinary anesthetic.

Pharmaceutical Compounds

The compounds used in the present methods include all hydrates,solvates, and complexes of the compounds. If a chiral center or anotherform of an isomeric center is present in a compound of the presentinvention, all forms of such isomer or isomers, including enantiomersand diastereomers, are intended to be covered herein. Compoundscontaining a chiral center may be used as a racemic mixture, anenantiomerically enriched mixture, or the racemic mixture may beseparated using well-known techniques and an individual enantiomer maybe used alone. The compounds described in the present invention are inracemic form or as individual enantiomers. The enantiomers can beseparated using known techniques, such as those described in Pure andApplied Chemistry 69, 1469-1474, (1997) IUPAC. In cases in whichcompounds have unsaturated carbon-carbon double bonds, both the cis (Z)and trans (E) isomers are within the scope of this invention. In caseswherein compounds may exist in tautomeric forms, such as keto-enoltautomers, each tautomeric form is contemplated as being included withinthis invention whether existing in equilibrium or predominantly in oneform.

When the structure of the compounds used in this invention includes anasymmetric carbon atom such compound can occur as racemates, racemicmixtures, and isolated single enantiomers. All such isomeric forms ofthese compounds are expressly included in this invention. Eachstereogenic carbon may be of the R or S configuration. It is to beunderstood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis, such as those described in“Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S.Wilen, Pub. John Wiley & Sons, N Y, 1981. For example, the resolutionmay be carried out by preparative chromatography on a chiral column.

The present disclosure is also intended to include use of all isotopesof atoms occurring on the compounds disclosed herein. Isotopes includethose atoms having the same atomic number but different mass numbers.Isotopically-labeled compounds can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Examples disclosed herein using an appropriateisotopically-labeled reagents in place of the non-labeled reagentsemployed.

The compounds of the instant invention may be in a salt form. As usedherein, a “salt” is a salt of the instant compound which has beenmodified by making acid or base, salts of the compounds. In the case ofcompounds used for treatment of mammals, the salt is pharmaceuticallyacceptable. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as phenols.The salts can be made using an organic or inorganic acid. Such acidsalts are chlorides, bromides, sulfates, nitrates, phosphates,sulfonates, formates, tartrates, maleates, malates, citrates, benzoates,salicylates, ascorbates, and the like. Phenolate salts are the alkalineearth metal salts, sodium, potassium or lithium. The term“pharmaceutically acceptable salt” in this respect, refers to therelatively non-toxic, inorganic and organic acid or base addition saltsof compounds of the present invention. These salts can be prepared insitu during the final isolation and purification of the compounds of theinvention, or by separately treating a purified compound of theinvention in its free base or free acid form with a suitable organic orinorganic acid or base, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

The present methods also encompass administering a physiologicallyfunctional derivative of the present compound. As used herein, the term“physiologically functional derivative” refers to a compound (e.g, adrug precursor) that is transformed in vivo to yield the presentcompound or its active metabolite, or a pharmaceutically acceptablesalt, hydrate or solvate of the compound. The transformation may occurby various mechanisms (e.g., by metabolic or chemical processes), suchas, for example, through hydrolysis in blood. Prodrugs are suchderivatives, and a discussion of the use of prodrugs is provided by T.Higuchi and W. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 ofthe A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987.

Dosages

In certain embodiments, the effective amount of the present compound isa dose of about 0.01 to about 3 mg of ketamine per kilogram of bodyweight of the subject (mg/kg), i.e., from about 0.01 mg/kg to about 3mg/kg body weight. In certain embodiments, the effective amount of thepresent compound ranges 0.001 to approximately 3 mg/kg body weight,0.001 to approximately 2 mg/kg body weight, from about 0.01 mg/kg toabout 3 mg/kg body weight, from about 0.01 to about 2 mg/kg of bodyweight, about 0.01 to about 1.5 mg/kg of body weight, about 0.05 toabout 1.4 mg/kg of body weight, about 0.05 to about 1.3 mg/kg of bodyweight, about 0.05 to about 1.2 mg/kg of body weight, about 0.05 toabout 1.1 mg/kg of body weight, about 0.01 to about 1 mg/kg of bodyweight, or about 0.05 to about 0.7 mg/kg of body weight. In someaspects, the dose is about 0.05 to about 0.5 mg/kg. In some aspects, thedose is less than about 0.5 mg/kg, less that about 0.4 mg/kg, or lessthan about 0.3 mg/kg body weight. In some aspects, the effective amountof the present compound is a dose in the range of from about 0.01 mg/kgto about 1.5 mg/kg body weight. In some aspects, the effective amount ofthe present compound is a dose in the range of from about 0.01 mg/kg toabout 1 mg/kg body weight. In some aspects, the effective amount of thepresent compound is a dose in the range of from about 0.01 mg/kg toabout 0.75 mg/kg body weight. In some aspects, the effective amount ofthe present compound is a dose in the range of from about 0.75 mg/kg toabout 1.5 mg/kg body weight. In some aspects, the effective amount ofthe present compound is a dose in the range of from about 0.5 mg/kg toabout 1.2 mg/kg body weight. In some aspects, the effective amount ofthe present compound is a dose in the range of from about 0.05 mg/kg toabout 0.5 mg/kg. In some aspects, the effective amount of the presentcompound is a dose of about 0.2 mg/kg or about 0.4 mg/kg body weight. Insome aspects, the dose of the present compound is, about 0.01 to about 1mg/kg, about 0.1 to about 0.5 mg/kg, about 0.8 to about 1.2 mg/kg, about0.7 to about 1.1 mg/kg, about 0.05 to about 0.7 mg/kg, about 0.01 mg/kg,about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg,about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg,about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg,about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg,about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg,about 2.0 mg/kg, or about 3 mg/kg body weight.

In certain embodiments, the dose of the present compound peradministration is from about 1 to about 250 mg, from about 10 mg toabout 300 mg, about 10 mg to about 250 mg, about 10 to about 200 mg,about 15 to about 175 mg, about 20 to about 175 mg, about 8 mg to about32 mg, about 50 mg to about 75 mg, about 25 to about 150 mg, about 25 toabout 125 mg, about 25 to about 100 mg, about 50 to about 100 mg, about50 mg to about 75 mg, about 75 mg to about 100 mg, or about 75 mg toabout 200 mg, about 1 mg, 2 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg,30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg,160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg,and 250 mg. In some aspects, the dose of the present compound is about50 mg. In some aspects, the dose of the present compound is about 75 mg.In some aspects, the total dose of the present compound is about 100 mg.

In some aspects, the therapeutically effective amount of the presentcompound is a sub-anesthetic dose. In some aspects, the therapeuticallyeffective amount of the present compound is a sub-analgesic dose. Incertain embodiments, the therapeutically effective amount of the presentcompound is below the level that results in one or more side effects ofthe compound. In certain embodiments, the therapeutically effectiveamount of the present compound is an anesthetic dose or analgesic dose.U.S. Patent Publication No. 20160067196.

In some aspects, the (therapeutically) effective amount of the presentcompound is about 0.01 mg to about 1000 mg, from about 0.01 mg to about500 mg, from about 0.1 mg to about 250 mg, or any amount or rangetherein. In another aspect, the (therapeutically) effective amount ofthe present compound is, e.g., 0.01 mg, 0.025 mg, 0.05 mg, 0.1 mg, 0.5mg, 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg,80 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg, or 500 mg.

In certain embodiments, a therapeutically effective dose of the presentcompound may be adjusted depending on conditions of the disease/disorderto be treated or prophetically treated, the age, body weight, generalhealth conditions, sex, and diet of the subject, dose intervals,administration routes, excretion rate, and combinations of drugs.

An initial dose of the present compound may be larger, followed by oneor more smaller maintenance doses. Other ranges are possible, dependingon the subject's response to the treatment. An initial dose may be thesame as, or lower or higher than subsequently administered doses.

The dose may be administered daily, weekly, biweekly, several timesdaily, semi-weekly, every other day, bi-weekly, quarterly, several timesper week, semi-weekly, monthly etc., to maintain an effective dosagelevel. The duration and frequency of treatment may depend upon thesubject's response to treatment.

In certain embodiments, a subject may be administered 1 dose, 2 doses, 3doses, 4 doses, 5 doses, 6 doses or more of the present composition. Incertain embodiments, a single dose of the present agent/composition isadministered in the present method. In certain embodiments, multipledoses of the present agent/composition (e.g., 2 doses, 3 doses, 4 doses,5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses or more) areadministered in the present method.

In certain embodiments, when there are more than one doses of thepresent compound/composition administered to a subject, the second doseis lower than the first dose. In certain embodiments, the second dose isan amount that is at most one-half, one-quarter, or one-tenth the amountof the first dose.

The number and frequency of doses may be determined based on thesubject's response to administration of the composition, e.g., if one ormore of the patient's symptoms improve and/or if the subject toleratesadministration of the composition without adverse reaction.

In certain embodiments, the present agent/composition is administered atleast once a day, at least twice a day, at least three times per day, ormore. In certain embodiments, the present agent/composition isadministered at least once a week, at least twice a week, at least threetimes per week, or more frequently. In certain embodiments, the presentagent/composition is administered at least twice per month, or at leastonce per month.

Treatment using the present method can continue as long as needed.

Dosing Time Frame

In certain embodiments, the present agent/composition is administered toa subject prior to a stressor. In certain embodiments, the presentagent/composition is administered to a subject both prior to and after astressor. In certain embodiments, the present agent/composition isadministered to a subject after a stressor. In certain embodiments, thepresent agent/composition is administered to a subject prior to astressor, and again prior to a recurrence of the stressor or a differentstressor.

In certain embodiments, the present agent/composition is administered tothe subject about 12 hours to about 4 weeks, about 18 hours to about 4weeks, about 1 day to about 3.5 weeks, about 2 days to about 3 weeks,about 3 days to about 3 weeks, about 4 days to about 3 weeks, about 5days to about 3 weeks, about 6 days to about 3 weeks, about 2 days toabout 2.5 weeks, about 3 days to about 2.5 weeks, about 4 days to about2.5 weeks, about 5 days to about 2.5 weeks, about 6 days to about 2.5weeks, about 1 week to about 2.5 weeks, about 1 week to about 2.5 weeks,about 1 week to about 2 weeks, about 3 days, about 4 days, about 5 days,about 6 days, about 1 week, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 2 weeks, about 2.5weeks, about 3 weeks, about 3.5 weeks, or about 4 weeks, prior to,and/or after a stressor.

In certain embodiments, the administration of the presentagent/composition is continued over a period of up to 2 days, up to 3days, up to 4 days, up to 5 days, up to 6 days, up to 1 week, up to 2weeks, up to 3 weeks, up to 4 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks,6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or longer.

In certain embodiments, the present agent/composition is administeredonce, twice, at least twice, at least three times, at least four times,at least five time, at least six times, at least seven times, at leasteight times, at least nine times, or more per treatment.

In certain embodiments, the present agent/composition is administered atleast once a day, at least twice a day, at least three times per day, atleast once a week, at least twice a week, at least three times a week,at least once per month, at least twice per month, or more frequently.Treatment can continue as long as needed.

Stressors

A stressor is a stimulus that causes stress. It can be an event or otherfactor that disrupts the body's homeostasis of temperature, bloodpressure, and/or other functions. In certain embodiments, a stressor isa traumatic or stressful event. Because humans have sophisticated brainsand thought processes, anticipating a disruption can also be a stressor.In certain embodiments, a stressor is injury, trauma, combat, warfare,surgery, an accident, a criminal assault, child abuse, natural orhuman-caused disasters, a crash, grief, hunger, heat, cold, chemicalexposure, chemical withdrawal, autoimmune disease, infectious disease,viral infection, cancer, exhaustion, physical distress, neuropathy,hyperalgesia, allodynia, emotional distress, or depression. A traumaticevent may be an event or something that threatens the person's life orthe life of a close one or it could be something witnessed.

A stressor may be acute, or may be chronic.

There are numerous physiological processes that are altered in responseto stress. Among these are altered cortisol, corticotropin,catecholamine and serotonin levels. These levels return to baselineafter an acute stressor is removed (McEwen N Eng J Med 1998338(3):171-179). These biochemical markers of stress in turn lead to illhealth and psychosocial disorders. Consequently, stress plays a majorrole in physical and mental health. Stress can affect the onset of, orsusceptibility to disease. It can also affect the progression or courseof disease even when there is another underlying pathophysiology of thedisease. Recovery from an existing disease can also be delayed due tostress. For example, stress is a contributing factor to high bloodpressure, heart disease, headaches, colitis, irritable bowel syndrome,temporo-mandibular joint disorder, cancer, peptic ulcers, insomnia, skindisorders and asthma. Stress can also aggravate other conditions such asmultiple sclerosis, diabetes, herpes, mental illness, substance abuseand psychiatric disorders characterized by the presence of violent oraggressive tendencies. Particularly, stress contributes to functionalsomatic disorders, affective disorders and major depressive disorder.These include disorders such as chronic fatigue syndrome (CFS),fibromyalgia (FMS), Gulf War Syndrome, anxiety and post-traumatic stressdisorder (PTSD). Stressors that disrupt normal exercise or sleeppatterns.

Additional examples of use include administration prior to militarydeployment to protect service members (active combat soldiers,battlefield surgeons, etc.) and even military working dogs againststress. Potential non-military use cases include, but are not limitedto: police, firefighters, first responders, EMTs, ER doctors, prisonguards (and prisoners), humanitarian aid workers, and refugees.

In certain embodiments, a subject may be administered the present agentor composition prior to a situation in which the subject (such as anearly responder or military personnel) is likely to be exposed totraumatic stress, immediately after exposure to traumatic stress, and/orwhen the subject feels that his or her PTSD symptoms are likely toappear.

Resilience to Stress

Resilience to stress refers to the capacity of a subject to adapt orchange successfully, and/or to maintain physiological, neurological, orpsychological homeostasis, in the face of a stressor (e.g., adversity).As used herein, the term “enhancing resilience” refers to increasing theability of a subject to experience a stressor (e.g., a traumatic event)without suffering a stress-induced affective disorder, and/or with lesspost-event symptomatology or disruption of homeostasis and/or normalactivities of daily living. In certain embodiments, improving resiliencecan prevent a stress-induced affective disorder. In certain embodiments,improving resilience can reduce at least one of the signs, symptoms, orsymptom clusters of a stress-induced affective disorder. In certainembodiments, the present method enhances a subject's resilience tostress, helps protect against developing stressor-relatedpsychopathology, decrease the functional consequences ofstressor-induced disorders (e.g., PTSD, etc.), and reduce medicalmorbidity and mortality.

The Connor-Davidson Resilience Scale (CD-RISC) is a 25-item self-reportscale, each rated on a 5-point scale (0-4), with higher scoresreflecting greater resilience (Connor K M & Davidson, J R T. Developmentof a new resilience scale: the Connor-Davidson Resilience Scale(CD-RISC). Depression and Anxiety, 2003: 18: 71-82).

Resilience, psychological growth and life satisfaction may be measuredwith the CD-RISC, the Purpose in Life Scale, the abbreviated MOS SocialSupport Survey, the PTGI, and the Q-LES-Q.

Combination Therapy

The present compound or composition may be administered to a subjectalone, or may be administered to a subject in combination with one ormore other treatments/agents.

In certain embodiments, the second agent is an anti-depressant, ananxiolytic, or combinations thereof. In certain embodiments, the secondagent is a serotonin reuptake inhibitor (SRI), or a selective serotoninreuptake inhibitor (SSRI). In certain embodiments, the second agent isfluoxetine, paroxetine, sertraline, lithium, riluzole, prazosin,lamotrigine, ifenprodil, or combinations thereof. In certainembodiments, the second agent is a dual serotonin norepinephrinereuptake inhibitor compound (DRI). In certain embodiments, the secondagent is venlafaxine, duloxetine, milnacipran, or combinations thereof.In certain embodiments, the second agent is a non-tricyclic triplereuptake inhibitor (TRI).

In certain embodiments, the present compound or composition isadministered to a subject in combination with one or moretreatments/agents such as antidepressants, analgesics, muscle relaxants,anorectics, stimulants, antiepileptic drugs, and sedative/hypnotics.Non-limiting examples of compounds that can be administered incombination with the present compound or composition include, neurontin,pregabalin, pramipexole, L-DOPA, amphetamine, tizanidine, clonidine,tramadol, morphine, tricyclic antidepressants, codeine, carbamazepine,sibutramine, amphetamine, valium, trazodone and combinations thereof.

In certain embodiments, combination therapy means simultaneousadministration of the compounds in the same dosage form, simultaneousadministration in separate dosage forms, or separate administration ofthe compounds.

In certain embodiments, the second agent/treatment is used as adjunctivetherapy to the present compound or composition. In certain embodiments,the treatment includes a phase wherein treatment with the secondagent/treatment takes place after treatment with the present compound orcomposition has ceased. In certain embodiments, the treatment includes aphase where treatment with the present compound or composition andtreatment with the second agent/treatment overlap.

Combination therapy can be sequential or can be administeredsimultaneously. In either case, these drugs and/or therapies are said tobe “co-administered.” It is to be understood that “co-administered” doesnot necessarily mean that the drugs and/or therapies are administered ina combined form (i.e., they may be administered separately (e.g., asseparate compositions or formulations) or together (e.g., in the sameformulation or composition) to the same or different sites at the sameor different times).

In certain embodiments, a subject is treated concurrently (orconcomitantly) with the present compound or composition and a secondagent. In certain embodiments, a subject is treated initially with thepresent compound or composition, followed by cessation of the presentcompound or composition treatment and initiation of treatment with asecond agent. In certain embodiments, the present compound orcomposition is used as an initial treatment, e.g., by administration ofone, two or three doses, and a second agent is administered to prolongthe effect of the present compound or composition, or alternatively, toboost the effect of the present compound or composition. A person ofordinary skill in the art will recognize that other variations of thepresented schemes are possible, e.g., initiating treatment of a subjectwith the present compound or composition, followed by a period whereinthe subject is treated with a second agent as adjunct therapy to thepresent compound or composition treatment, followed by cessation of thepresent compound or composition treatment.

The present compound and the other pharmaceutically active agent(s) maybe administered together or separately and, when administered separatelythis may occur simultaneously or sequentially in any order. The amountsof the present compound and the other pharmaceutically active agent(s)and the relative timings of administration will be selected in order toachieve the desired combined therapeutic effect.

In various embodiments, the therapies (e.g., a composition providedherein and a second agent in a combination therapy) are administeredless than 5 minutes apart, less than 30 minutes apart, 1 hour apart, atabout 1 hour apart, at about 1 to about 2 hours apart, at about 2 hoursto about 3 hours apart, at about 3 hours to about 4 hours apart, atabout 4 hours to about 5 hours apart, at about 5 hours to about 6 hoursapart, at about 6 hours to about 7 hours apart, at about 7 hours toabout 8 hours apart, at about 8 hours to about 9 hours apart, at about 9hours to about 10 hours apart, at about 10 hours to about 11 hoursapart, at about 11 hours to about 12 hours apart, at about 12 hours to18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart,36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84hours to 96 hours apart, or 96 hours to 120 hours part. In certainembodiments, the therapies are administered no more than 24 hours apartor no more than 48 hours apart. In certain embodiments, two or moretherapies are administered within the same patient visit. In otherembodiments, the composition provided herein and the second agent areadministered concurrently. In other embodiments, the compositionprovided herein and the second agent are administered at about 2 to 4days apart, at about 4 to 6 days apart, at about 1 week part, at about 1to 2 weeks apart, or more than 2 weeks apart. In certain embodiments,administration of the same agent may be repeated and the administrationsmay be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. Inother embodiments, administration of the same agent may be repeated andthe administration may be separated by at least at least 1 day, 2 days,3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In certain embodiments, a composition providedherein and a second agent are administered to a subject in a sequenceand within a time interval such that the composition provided herein canact together with the other agent to provide an increased benefit thanif they were administered otherwise. For example, the second activeagent can be administered at the same time or sequentially in any orderat different points in time; however, if not administered at the sametime, they should be administered sufficiently close in time so as toprovide the desired therapeutic or prophylactic effect. In oneembodiment, the composition provided herein and the second active agentexerts their effect at times which overlap. Each second active agent canbe administered separately, in any appropriate form and by any suitableroute. In other embodiments, the composition provided herein isadministered before, concurrently or after administration of the secondactive agent. The term “about” refers to +10% of the referenced value.In other embodiments, courses of treatment are administered concurrentlyto a patient, i.e., individual doses of the second agent areadministered separately yet within a time interval such that thecompound provided herein can work together with the second active agent.For example, one component can be administered once per week incombination with the other components that can be administered onceevery two weeks or once every three weeks. In other words, the dosingregimens are carried out concurrently even if the therapeutics are notadministered simultaneously or during the same day. The second agent canact additively or synergistically with the compound provided herein. Inone embodiment, the composition provided herein is administeredconcurrently with one or more second agents in the same pharmaceuticalcomposition. In another embodiment, a composition provided herein isadministered concurrently with one or more second agents in separatepharmaceutical compositions. In still another embodiment, a compositionprovided herein is administered prior to or subsequent to administrationof a second agent. Also contemplated are administration of a compositionprovided herein and a second agent by the same or different routes ofadministration, e.g., oral and parenteral. In certain embodiments, whenthe composition provided herein is administered concurrently with asecond agent that potentially produces adverse side effects including,but not limited to, toxicity, the second active agent can advantageouslybe administered at a dose that falls below the threshold that theadverse side effect is elicited.

Encompassed by the present disclosure are methods to prophylacticallytreat a subject prior to a stressor. In certain embodiments, the presentmethod prevents or delays a stress-induced affective disorder orstress-induced psychopathology in a subject. In certain embodiments,stress-induced affective disorders include major depressive disorder andposttraumatic stress disorder.

Stress-Induced Affective Disorders

There are numerous disorders that are either caused by or exacerbated bystress. These include addictive disorders such as substance abuse,anorexia, bulimia, obesity, smoking addiction, and weight addiction;anxiety disorders such as agoraphobia, anxiety disorder, obsessivecompulsive disorder, panic attacks, performance anxiety, phobias, andpost-traumatic stress disorder; autoimmune diseases such as allergies,arthritis, fibromyalgia, fibromytosis, lupus, multiple sclerosis,rheumatoid arthritis, Sjogren's syndrome, and vitiligo; cancer such asbone cancer, brain cancer, breast cancer, cervical cancer, colon cancer,Hodgkin's disease, leukemia, liver cancer, lung cancer, lymphoma,multiple myeloma, ovarian cancer, pancreatic cancer, and prostatecancer; cardiovascular disorders such as arrhythmia, arteriosclerosis,Burger's disease, essential hypertension, fibrillation, mitral valveprolapse, palpitations, peripheral vascular disease, Raynaud's disease,stroke, tachycardia, and Wolff-Parkinson-White Syndrome; anddevelopmental disorders such as attention deficit disorder,concentration problems, conduct disorder, dyslexia, hyperkinesis,language and speech disorders, and learning disabilities.

Anxiety Disorders

The five major types of anxiety disorders are: panic disorder,obsessive-compulsive disorder, post-traumatic stress disorder,generalized anxiety disorder and phobias (including social phobia, alsocalled social anxiety disorder). Each anxiety disorder has its owndistinct features, but they are all bound together by the common themeof excessive, irrational fear and dread. It is common for an anxietydisorder to accompany depression, eating disorders, substance abuse, oranother anxiety disorder.

Panic disorder is characterized by repeated episodes of intense fearthat strike often and without warning. Physical symptoms include chestpain, heart palpitations, shortness of breath, dizziness, abdominaldistress, feelings of unreality, and fear of dying. Obsessive-compulsivedisorder is characterized by repeated, unwanted thoughts or compulsivebehaviors that seem impossible to stop or control. Generalized AnxietyDisorder is characterized by exaggerated worrisome thoughts and tensionabout everyday routine life events and activities, lasting at least sixmonths. Almost always anticipating the worst even though there is littlereason to expect it; accompanied by physical symptoms, such as fatigue,trembling, muscle tension, headache, or nausea. Phobias arecharacterized into two major types of phobias, social phobia andspecific phobia. People with social phobia have an overwhelming anddisabling fear of scrutiny, embarrassment, or humiliation in socialsituations, which leads to avoidance of many potentially pleasurable andmeaningful activities. People with specific phobia experience extreme,disabling, and irrational fear of something that poses little or noactual danger; the fear leads to avoidance of objects or situations andcan cause people to limit their lives unnecessarily.

Posttraumatic Stress Disorder (PTSD)

Typically, a subject suffering from PTSD was exposed to a traumaticevent in which the person experienced, witnessed, or was confronted withan event or events that involved actual or threatened death or seriousinjury, or a threat to the physical integrity of self or others and theperson's response involved intense fear, helplessness, or horror.

Having repeated intrusive memories of the trauma exposure is one of thecore symptoms of PTSD. Patients with PTSD are known to displayimpairments in learning and memory during neuropsychological testing.Other core symptoms of PTSD include heightened stress sensitivity(startle), tension and anxiety, memory disturbances, and dissociation.

In certain embodiments, the present method prevents or inhibits thedevelopment of post-traumatic stress disorder (PTSD) in a subject. Incertain embodiments, the present method prevents or inhibits thedevelopment of one or more PTSD-like symptoms. In certain embodiments, asubject may be administered the present agent or composition prior to asituation in which the subject (such as an early responder or militarypersonnel) is likely to be exposed to traumatic stress, immediatelyafter exposure to traumatic stress, and/or when the subject feels thathis or her PTSD symptoms are likely to appear.

Typically, the traumatic event is persistently re-experienced in one ormore of the following ways: recurrent and intrusive distressingrecollections of the event, including images, thoughts, or perceptions,recurrent distressing dreams of the event, acting or feeling as if thetraumatic event were recurring (includes a sense of reliving theexperience, illusions, hallucinations, and dissociative flashbackepisodes, including those that occur on awakening or when intoxicated),intense psychological distress at exposure to internal or external cuesthat symbolize or resemble an aspect of the traumatic event,physiological reactivity on exposure to internal or external cues thatsymbolize or resemble an aspect of the traumatic event. An individualsuffering from PTSD also has persistent avoidance of stimuli associatedwith the trauma and numbing of general responsiveness (not presentbefore the trauma), as indicated by 3 or more of the following: effortsto avoid thoughts, feelings, or conversations associated with thetrauma, efforts to avoid activities, places, or people that arouserecollections of the trauma, inability to recall an important aspect ofthe trauma, significantly diminished interest or participation insignificant activities, feeling of detachment or estrangement fromothers, restricted range of affect (e.g., unable to have lovingfeelings), sense of a foreshortened future (e.g., does not expect tohave a career, marriage, children, or a normal life span), persistentsymptoms of increased arousal (not present before the trauma), asindicated by 2 or more of the following: difficulty falling or stayingasleep, irritability or outbursts of anger, difficulty concentrating,hypervigilance, exaggerated startle response. The disturbance, which haslasted for at least a month, causes clinically significant distress orimpairment in social, occupational, or other important areas offunctioning.

In certain embodiments, the present compound or composition prevents,reduces, eliminates or delays one or more of the symptoms including, butnot limited to, re-experiencing of the traumatic experience in the formof intrusive memories, nightmares, flashbacks; emotional and physicalreactions triggered by reminders of the trauma; distancing from others;decreased interest in activities and other people; numbing of feelings;avoidance of trauma reminders; hyperarousal symptoms, includingdisrupted sleep, irritability, hypervigilance, decreased concentration;increased startle reflex; and combinations thereof.

Whatever the source of the problem, some people with PTSD repeatedlyrelive the trauma in the form of nightmares and disturbing recollectionsduring the day. They may also experience other sleep problems, feeldetached or numb, or be easily startled. They may lose interest inthings they used to enjoy and have trouble feeling affectionate. Theymay feel irritable, more aggressive than before, or even violent. Thingsthat remind them of the trauma may be very distressing, which could leadthem to avoid certain places or situations that bring back thosememories.

The disorder may be accompanied by depression, substance abuse, or oneor more other anxiety disorders. In severe cases, the person may havetrouble working or socializing.

Major Depressive Disorder

Major depressive disorder refers to a class of syndromes characterizedby negative affect and repeated episodes of depression without anyhistory of independent episodes of mood elevation and over-activity thatfulfill the criteria of mania. Multiple subtypes of major depressivedisorders are recognized, including these with atypical characteristics,psychotic components, etc. The age of onset and the severity, durationand frequency of the episodes of depression are all highly variable. Thedisorder may begin at any age. The symptoms of major depressive disordertypically develop over days to weeks. Prodromal symptoms includegeneralized anxiety, panic attacks, phobias or depressive symptoms andmay occur during several months preceding the episode. Individualepisodes also last between 3 and 12 months but recur less frequently.Most patients are asymptomatic between episodes, but a minority ofpatients may develop a persistent depression, mainly in old age.Individual episodes of any severity are often precipitated by stressfullife events. Common symptoms of a depressive episode include reducedconcentration and attention; reduced self-esteem and self-confidence;ideas of guilt and unworthiness, ideas or acts of self-harm or suicide;disturbed sleep; and diminished appetite. In certain embodiments, amajor depressive episode follows a psychosocial stressor, e.g., death ofa loved one, marital separation, childbirth or the end of an importantrelationship. The lowered mood varies little from day to day and isoften unresponsive to circumstances, yet may show a characteristicdiurnal variation as the day goes on. As with manic episodes, theclinical presentation shows marked individual variations, and atypicalpresentations are particularly common in adolescence. In some cases,anxiety, distress, and motor agitation may be more prominent at timesthat the depression, and the mood change may also be masked by addedfeatures such as irritability, excessive consumption of alcohol,histrionic behavior, and exacerbation of pre-existing phobic orobsessional symptoms, or by hypochondria.

Psychiatric Evaluations

The psychiatric conditions of the subject, and/or the effects orefficacy of treatment with the present agent/composition, may beevaluated by the subject and/or a medical professional, e.g., thesubject's physician. In certain embodiments, the evaluation is conductedwithin about 10 minutes, within about 15 minutes, within about 20minutes, within about 25 minutes, within about 0.5 hours, within about 1hour, within about 2 hours, within about 2.5 hours, within about 3hours, within about 3.5 hours, within about 4 hours, within about 4.5hours, within about 5 hours, within about 5.5 hours, within about 6hours, within about 6.5 hours, within about 7 hours, within about 7.5hours, within about 8 hours, within about 8.5 hours, within about 9hours, within about 9.5 hours, within about 10 hours, within about 10.5hours, within about 11 hours, within about 11.5 hours, within about 12hours, within about 18 hours, within about 1 day, within about 2 days,within about 3 days, within about 4 days, within about 5 days, withinabout 6 days, within about 1 week, within about 2 weeks, within about 3weeks, within about 4 weeks, within about 1 month, within about 2months, within about 3 months, within about 4 months, within about 5months, within about 6 months, within about 1 year, within about 2years, or longer, before, during, or after a stressor and/oradministration of the present agent/composition.

Psychiatric evaluations of a patient being treated with the presentmethod can be conducted to determine whether the method is effective. Incertain embodiments, the psychiatric evaluation may be carried outbefore treatment, at the time of treatment, during treatment, and/orafter treatment. When the psychiatric evaluation is carried out bothbefore treatment and after (and/or during) treatment with the presentmethod, the results of the evaluation before treatment can provide abaseline for comparison to the results of the evaluation during and/orafter treatment. In certain embodiments, psychiatric evaluation isconducted only after treatment.

Psychophysiological stress tests can be performed to measure the amountof stress-induced anxiety present in the various systems of the body(i.e. muscular, cardiovascular, digestive, respiratory and neurologicalsystems). These stress tests are routinely used in the art. Test resultsare compared to both local and national norms, to determine if theindividual is exhibiting an excessive amount of physiological anxietyand whether or not they are able to recover from a standardizedstressful stimuli in an appropriate length of time.

Psychiatric testing can be used to monitor a subject to determine theemotional and/or social etiology of the stress disorder. These tests areknown in the art and include health-related assessments, mental healthassessments, personality tests, and personality type assessment.

In certain embodiments, clinician-administered evaluation and/orself-report instruments are used, with the aim of measuring baselinesymptomatology as well as drug actions on (1) the overall severity ofthe disorder, (2) the core symptoms, and (3) depressed mood.

Non-limiting examples of psychiatric evaluation tools and questionnairesinclude the following measures.

The Diagnostic and Statistical Manual of Mental Disorders (DSM-5)includes the revised diagnostic criteria for PTSD. See, AmericanPsychiatric Association: Diagnostic and Statistical Manual of MentalDisorders, Fifth Edition. Arlington, Va., American PsychiatricAssociation, 2013. See alsoptsd.va.gov/professional/PTSD-verview/dsm5_criteria_ptsd.asp.

The Structured Clinical Interview for DSM-IV Axis I Disorders, PatientEdition (SCID-P) is a semi-structured interview that provides probequestions as well as follow-up questions to be asked by the clinician toassist in diagnosis. First et al., Structured Clinical Interview forDSM-IV TR Axis I Disorders, Research Version, Patient Edition(SCID-I/P). New York: New York State Psychiatric Institute, BiometricsResearch; 2001. It includes an overview to obtain information aboutdemographics, work, chief complaint, history of present illness, pasthistory, treatment history, and current functioning. The main body ofSCID-P includes 9 modules that are designed to diagnose 51 mentalillnesses in all.

The SCID-P for DSM-5 is the SCID—Patient version, and is the nextedition of the SCID modified to incorporate the new DSM-5 criteria.

The Clinician-Administered PTSD Scale (CAPS) is a structured clinicalinterview designed to assess the essential features of PTSD as definedby the DSM-IV. Weathers et al., Clinician-administered PTSD scale: areview of the first ten years of research. Depress Anxiety. 2001;13(3):132-156. The CAPS can be used to provide categorical ratings ofdiagnostic status as well as a quantitative index of symptom severity.Both frequency and intensity scores are derived for each individualsymptom. The CAPS total score is based on an individual's response tothe 17 items that assess the frequency and intensity of current PTSDsymptoms. Subscales of the CAPS are utilized to assess specific symptomclusters. The total score can range from 0 to 136.

The Clinician-Administered PTSD Scale for DSM-5 (CAPS-5) is a 30-itemstructured interview that can be used to make current (past month)diagnosis of PTSD, make lifetime diagnosis of PTSD, and to assess PTSDsymptoms over the past week. CAPS-5 is a 30-item questionnaire,corresponding to the DSM-5 diagnosis for PTSD. The language of theCAPS-5 reflects both changes to existing symptoms and the addition ofnew symptoms in DSM-5. Weathers, F. W., et al (2013). TheClinician-Administered PTSD Scale for DSM-5 (CAPS-5).

The Treatment Outcome PTSD Scale (TOP-8) is a briefinterviewer-administered scale designed specifically for the assessmentof commonly occurring signs and symptoms of PTSD that are subject tochange in response to treatment (Davidson, J. R., & Colket, J. T.(1997). The eight-item treatment-outcome post-traumatic stress disorderscale: A brief measure to assess treatment outcome in post-traumaticstress disorder. International Clinical Psychopharmacology, 12(1),41-45). The TOP-8 is comprised of eight items, each measured on a scaleof 0-4, with defined anchors given for each item. The items arerepresentative of the three core features of PTSD with a maximumpossible score of 32.

The Hamilton Psychiatric Rating Scale for Anxiety (HAM-A) is a widelyused observational rating measure of anxiety severity. The scaleconsists of 14 items. Each item is rated on a scale of 0 to 4. Thisscale is administered to assess the severity of anxiety and itsimprovement during the course of treatment. The HAM-A total score is thesum of the 14 items and the score ranges from 0 to 56. Hamilton M. TheAssessment of Anxiety-States by Rating. Br J Med Psychol. 1959;32(1):50-55.

The Montgomery-Asberg Depression Rating Scale (MADRS) is a 10-iteminstrument used for the evaluation of depressive symptoms in adults andfor the assessment of any changes to those symptoms. Montgomery S. A.,et al., A new depression scale designed to be sensitive to change. Br JPsychiatry. 1979 April; 134:382-389. Each of the 10 items is rated on ascale of 0 to 6, with differing descriptors for each item. Theseindividual item scores are added together to form a total score, whichcan range between 0 and 60 points.

The Young Mania Rating Scale, item 1 (YMRS-1) used to assess moodelevation on the infusion days. Young R C, et al. Rating-Scale forMania—Reliability, Validity and Sensitivity. Br J Psychiatry. 1978;133(November):429-435.

The Brief Psychiatric Rating Scale (BPRS) is used to assess acutebehavioral changes during the infusions. Overall J E et al., The BriefPsychiatric Rating-Scale. Psychol. Rep. 1962; 10(3):799-812 Four keyBPRS items for the positive (+) symptoms of psychosis are used:conceptual disorganization, hallucinatory behavior, suspiciousness, andunusual thought content. Three items representing the negative (−)symptoms of psychosis will also be used: blunted affect, emotionalwithdrawal, and motor retardation.

The Clinician-Administered Dissociative States Scale (CADSS) is used tomeasure dissociative effects during the infusions. Bremner J D, et al.,Measurement of Dissociative States with the Clinician-AdministeredDissociative States Scale (CADSS). J Trauma Stress. 1998; 11(1):125-136The scale includes 19 questions and 8 observer ratings scored from 0(not at all) to 4 (extremely). The CADSS measures impairment in bodyperception, environmental perception, time perception, memoryimpairment, and feelings of unreality.

The Patient Rating Inventory of Side Effects (PRISE) is a patientself-report used to qualify side effects by identifying and evaluatingthe tolerability of each symptom. Levine J, Schooler N R. SAFTEE: Atechnique for the systematic assessment of side effects in clinicaltrials. Psychopharmacol Bull. 1986; 22(2):343-381.

The Clinical Global Impression (CGI) scale assesses treatment responsein psychiatric patients. The administration time is 2 minutes. Thisscale consists of three items: Severity of Illness (item 1); GlobalImprovement (item 2); and Efficacy Index (item 3). Item 1 is rated on aseven-point scale (1=normal, 7=among the most extremely ill patients) asis item 2 (1=very much improved, 7=very much worse). Each includes anadditional response of “not assessed.” Item 3 is rated on a four-pointscale (from “none” to “outweighs therapeutic effect”).

The Impact of Events Scale (IES) is one of the most widely usedself-report measures of stress reactions to traumatic events. Horowitzet al., Impact of Event Scale: a measure of subjective stress. PsychosomMed. 1979 May; 41(3):209-218. See also, Weiss et al., The Impact ofEvent Scale—Revised In: Wilson J, Keane T M, eds. Assessingpsychological trauma and PTSD. New York: Guilford; 1996:399-411. Itmeasures both intrusion and avoidance. Sundin et al., Impact of EventScale: psychometric properties. Br J Psychiatry. 2002 March;180:205-209. Joseph S. Psychometric evaluation of Horowitz's Impact ofEvent Scale: a review. J Trauma Stress. 2000 January; 13(1):101-113. Thetotal score can range from 0 to 75.

The Posttraumatic Stress Disorder Checklist (PCL-5) is a 17-itemself-report measure reflecting DSM-5 symptoms of PTSD. The PCL-5measures symptoms in response to stressful situations (Weathers, F., etal. (1993). The PTSD checklist (PCL): Reliability, validity, anddiagnostic utility. Annual Convention of the International Society forTraumatic Stress Studies, San Antonio, Tex.).

The Quick Inventory of Depressive Symptomatology, Self Report (QIDS-SR)is a 16-item self-rated instrument designed to assess the severity ofdepressive symptoms present in the past seven days. Rush A J, Trivedi MH, Ibrahim H M et al. The 16-Item quick inventory of depressivesymptomatology (QIDS), clinician rating (QIDS-C), and self-report(QIDS-SR): a psychometric evaluation in patients with chronic majordepression. Biol. Psychiatry. 2003; 54(5):573-583. The 16 items coverthe nine symptom domains of major depression, and are rated on a scaleof 0-3. Total score ranges from 0 to 27, with ranges of 0-5 (normal),6-10 (mild), 11-15 (moderate), 16-20 (moderate to severe), and21+(severe).

The Childhood Trauma Questionnaire (CTQ) is a 28-item self-reportinstrument that assesses childhood trauma in the following areas:physical, sexual and emotional abuse and physical and emotional neglect.Bernstein D P, Stein J A, Newcomb M D et al. Development and validationof a brief screening version of the Childhood Trauma Questionnaire.Child Abuse Negl. 2003 February; 27(2): 169-190. Each item is rated on ascale of 1 (never true) to 5 (very often true). The 5 subscales are thentotaled, with scores ranging from 5-25 for each traumatic category.

Visual Analogue Scales (VAS) are used to assess subjective statechanges. Bond A, Lader M. The use of analogue scales in ratingsubjective feelings. Br J Med Psychol. 1974; 47(3):211-218. They are100-mm horizontal lines marked proportionately to the perceivedintensity of the subjective experience (0=not at all, to 10=extremely)for the following states: anxious, depressed, drowsy, high, hungry, andnauseous.

The Sheehan Disability Scale (SDS) is a self-report disability measure.It has demonstrated sensitivity to impairment and changes as a result oftreatment across a wide range of psychiatric disorders. The SDS asksonly about current levels of impairment, providing no indication ofwhether the person has done better or worse in the past, thus making ita reasonable short-term outcome measure that is un-confounded byhistorical impressions. The dependent variable is the total score, whichis based on the sum of three 10-point items (work, social life, andfamily life), with higher scores reflecting greater disability. SheehanD. The Anxiety Disease. New York, N.Y.: Scribner; 1983.

The Wechsler Abbreviated Scale of Intelligence 2-Subtest (WASI-2) is areliable brief measure of IQ for 6 to 89 year-olds that includesVocabulary (an estimate of verbal fluid abilities) and Matrix Reasoning(an estimate of nonverbal fluid abilities). Wechsler D. WechslerAbbreviated Scale of Intelligence San Antonio, Tex.: PsychologicalCorporation; 1999. It is extensively used in clinical, educational, andresearch settings. Average reliability coefficient is 0.96 andtest-retest reliability is 0.88.

The Hopkins Verbal Learning Test (HVLT) is a repeatable test of memoryacquisition and delayed recall of words. Subjects are presented with thesame 12-item list for 3 learning trials and asked each time to repeatthe items on each list. Delayed recall and recognition conditions areadministered later. Dependent variables used in this study include totallearning over the 3 trials (for the acquisition variable) and totaldelayed recall score (for the recall component). Brandt J, Benedict R.Hopkins Verbal Learning Test, Revised. Odessa, Fla.: PsychologicalAssessment Resources; 1997.

The Profile of Mood States-Bipolar (POMS-Bi) scale measures moods andfeelings primarily in clinical rather than nonclinical settings. It canhelp to determine an individual's psychiatric status for therapy, or beused to compare mood profiles associated with various personalitydisorders. It is also a useful instrument in identifying the effects ofdrug treatments.

The Post-Traumatic Cognitions Inventory (PTCI) is a 33-item scale, whichis rated on a Likert-type scale ranging from 1 (totally disagree) to 7(totally agree). Scale scores are formed for the three subscales, whichshow a high degree of intercorrelation (rs=0.57-0.75).

The New Cognitions scale is a 6-item pilot scale, which is rated on aLikert-type scale ranging from 1 (not at all) to 4 (a lot). The scale isbased on the Post Traumatic Growth Inventory (PTGI) from which itemshave been directly selected (new items were added to the scale as well),and on the Brief-COPE (see Carver, C. S. (1997) “You want to measurecoping but your protocol's too long: Consider the brief COPE.”International Journal of Behavioral Medicine 4; 92-100).

The Medical Outcomes Study (MOS) Social Support Survey is a 19-itemself-report measure designed to assess levels of functional socialsupport. The MOS-SS has two subscales (emotional and instrumental socialsupport) to identify potential social support deficits (Sherbourne, C.D. & Stewart, A. L. (1991). “The MOS Social Support Survey.” Soc Sci Med32(6): 705-714).

The Purpose in Life test-Short Form (PIL-SF) is a brief, 4-item form ofthe 20-item Purpose in Life test. This scale asks respondents to reportto what extent they have achieved their goals in life, and to whatextent they perceive their life to be meaningful or purposeful.(Schulenberg et al 2010; Psychotherapy (Chic). 2008 December;45(4):447-63).

Posttraumatic Growth Inventory (PTGI)-Short Version is a 10-itemshortened version of the PTGI self-report questionnaire (ref). It asksrespondents to rate the extent to which they have changed as the resultof experiencing a highly stressful life event. Items span positivechanges in five domains: relating to others, new possibilities, personalstrength, spiritual change, and appreciation of life (Cann, A., et al.(2010). A short form of the Posttraumatic Growth Inventory. Anxiety,Stress & Coping, 23, 127-137).

The Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q)is a self-report scale measuring the degree of enjoyment andsatisfaction experienced by subjects in various areas of dailyfunctioning. The summary scores are reliable and valid measures of thesedimensions in a group of depressed subjects (Endicott J, et al. Qualityof Life Enjoyment and Satisfaction Questionnaire: A New Measure.Psychopharmacology Bulletin; 1993; 29:321-326).

In certain embodiments, self-evaluation of the subject being treated isconducted.

Pharmaceutical Compositions

While it is possible that the present a compound, as well as salts,solvates and physiological functional derivatives thereof, may beadministered as the raw chemical, it is possible to present the activeingredient as a pharmaceutical composition. Accordingly, the inventionfurther provides a pharmaceutical composition, which comprises thepresent compound and/or salts, solvates and physiological functionalderivatives thereof, and one or more pharmaceutically acceptablecarriers, diluents, or excipients. The carrier(s), diluent(s) orexcipient(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof. In accordance with another aspect of the inventionthere is also provided a process for the preparation of a pharmaceuticalcomposition including admixing the present compound, or salts, solvatesand physiological functional derivatives thereof, with one or morepharmaceutically acceptable carriers, diluents or excipients.

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) (pharmaceutically acceptable excipients) that make up thecarrier, as well as any product which results, directly or indirectly,from combination, complexation or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the pharmaceutical compositions of the presentinvention encompass any composition made by admixing compound 20, andpharmaceutically acceptable excipients.

Acceptable excipients, diluents, and carriers for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington: The Science and Practice of Pharmacy. Lippincott Williams &Wilkins (A. R. Gennaro edit. 2005). The choice of pharmaceuticalexcipient, diluent, and carrier can be selected with regard to theintended route of administration and standard pharmaceutical practice.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are “generally regarded assafe”, e.g., that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopoeia or other generally recognized pharmacopeias for usein animals, and more particularly in humans.

Pharmaceutical compositions of the present invention may be presented inunit dose forms containing a predetermined amount of active ingredientper unit dose. Such a unit may contain, for example, 5 μg to 1 g,preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of the presentcompound, depending on the condition being treated, the route ofadministration and the age, weight and condition of the patient. Suchunit doses may therefore be administered more than once a day. Preferredunit dosage compositions are those containing a daily dose or sub-dose(for administration more than once a day), as herein above recited, oran appropriate fraction thereof, of an active ingredient. Furthermore,such pharmaceutical compositions may be prepared by any of the methodswell known in the pharmacy art.

Pharmaceutical compositions of the present invention may be adapted foradministration by any appropriate route, for example by the oral(including buccal or sublingual), inhaled, nasal, ocular, or parenteral(including intravenous and intramuscular) route. The present compositionmay be injected. Such compositions may be prepared by any method knownin the art of pharmacy, for example by bringing into association theactive ingredient with the carrier(s) or excipient(s).

In a further embodiment, the present invention provides a pharmaceuticalcomposition adapted for administration by the oral route, the treatmentof stress-induced affective disorder.

Pharmaceutical compositions of the present invention which are adaptedfor oral administration may be presented as discrete units such ascapsules or tablets; powders or granules; solutions or suspensions inaqueous or non-aqueous liquids; edible foams or whips; or oil-in-waterliquid emulsions or water-in-oil liquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing and coloringagent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

It should be understood that, in addition to the ingredientsparticularly mentioned above, the compositions may include other agentsconventional in the art having regard to the type of formulation inquestion, for example those suitable for oral administration may includeflavoring agents.

A therapeutically effective amount of a compound of the presentinvention will depend upon a number of factors including, for example,the age and weight of the subject, the precise condition requiringtreatment and its severity, the nature of the formulation, and the routeof administration, and will ultimately be at the discretion of theattendant physician or veterinarian.

Kits

Another aspect of the disclosure is a kit containing a reagent orreagents for measuring at least one biomarker in a biological sample,instructions for measuring the at least one biomarker, and/orinstructions for evaluating or monitoring efficacy of a prophylactictreatment in a patient based on the level of the at least one biomarker,and/or instructions for assessing a prophylactic therapy in a patient.In some embodiments, the kit contains reagents for measuring from 1 toabout 20 biomarkers, including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more biomarkers as describedherein.

The invention may also encompass arrays, microarrays, chips, biochips,etc.

Any of the compositions described herein may be comprised in a kit. Inone embodiment, the kit contains a reagent for measuring at least onebiomarker in a biological sample, instructions for measuring the atleast one biomarker, and instructions for evaluating or monitoringefficacy of a prophylactic treatment in a patient based on the level ofthe at least one biomarker. In some embodiments, the kit containsreagents for measuring the level of at least 2, 3, 4, 5, 6 or 10 (ormore) biomarkers. The kit may also be customized for determining theefficacy of therapy for a stress-induced affective disorder orstress-induced psychopathology, and thus provides the reagents fordetermining 50 or fewer, 40 or fewer, 30 or fewer, or 25 or fewerbiomarkers.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed(e.g., sterile, pharmaceutically acceptable buffer and/or otherdiluents). However, various combinations of components may be comprisedin a vial. The kits also will typically include a means for containingthe reagents, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution may be an aqueous solution. Thecomponents of the kit may also be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

Such kits may also include components that preserve or maintain thereagents or that protect against their degradation. Such components maybe protease inhibitors or protect against proteases. Such kits generallywill comprise, in suitable means, distinct containers for eachindividual reagent or solution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

This invention will be better understood from the examples, whichfollow. However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims that follow thereafter.

Example 1 Prophylactic Ketamine Alters Nucleotide and NeurotransmitterMetabolism in Brain and Plasma Following Stress

Recently, we have shown that ketamine given prior to stress exposureprotects against the development of depressive-like behavior in mice.These data suggest that it may be possible to prevent the induction ofaffective disorders before they develop by administering prophylacticpharmaceuticals, a relatively nascent and unexplored strategy forpsychiatry. Here, we performed metabolomics analysis of brain and plasmafollowing prophylactic ketamine treatment in order to identify markersof stress resilience enhancement. We administered prophylactic ketaminein mice to buffer against fear expression. Following behavioralanalyses, untargeted metabolomic profiling was performed on bothhemispheres of the prefrontal cortex (PFC) and the hippocampus (HPC),and plasma. We found that prophylactic ketamine attenuated learned fear.Eight metabolites were changed in the PFC and HPC upon ketaminetreatment. Purine and pyrimidine metabolism were most significantlychanged in the HPC, PFC, and, interestingly, plasma of mice two weeksafter prophylactic administration. Moreover, most precursors toinhibitory neurotransmitters were increased whereas precursors toexcitatory neurotransmitters were decreased. Strikingly, these long-termmetabolomic changes were not observed when no stressor was administered.Our results suggest that prophylactic treatment differentially affectspurine and pyrimidine metabolism and neurotransmission in brain andplasma following stress, which may underlie the long-lasting resilienceto stress induced by a single injection of ketamine. These data mayprovide novel targets for prophylactic development, and indicate aninteraction effect of prophylactic ketamine and stress. To ourknowledge, this is the first study that identifies metabolomicalterations and biomarker candidates for prophylactic ketamine efficacyin mice.

Here, in order to begin to address the mechanism behind the efficacy ofprophylactic treatment, we administered saline or ketamine (30 mg kg⁻¹)1 week before a 3-shock contextual fear-conditioning (CFC) paradigm orbefore context exposure without shocks, and subsequently profiled themetabolome of both hemispheres of the PFC and HPC, as well as plasma. Aswe previously reported (McGowan et al, 2017), prophylactic ketamineattenuated learned fear. Purine and pyrimidine metabolism wassignificantly altered in the HPC, PFC, and plasma following prophylacticketamine treatment and stress as compared with saline-treated mice.Moreover, most precursors to inhibitory neurotransmitters wereincreased, while most precursors to excitatory neurotransmitters weredecreased. Interestingly, these changes were not observed innon-stressed mice, indicating that ketamine interacts with a stressfulexperience to induce long-lasting changes in the metabolome and onbehavior. These results point to potential metabolites as mediators inketamine's resilience-enhancing effects, which may be easilyidentifiable in the clinic.

Materials and Methods Mice

129S6/SvEvTac mice were purchased from Taconic (Hudson, N.Y.) at 7 weeksof age. Mice were housed 4-5 per cage in a 12-h (06:00-18:00) light-darkcolony room at 22° C. Food and water were provided ad libitum.Behavioral testing was performed during the light phase. All experimentswere approved by the Institutional Animal Care and Use Committee at NewYork State Psychiatric Institute (NYSPI).

Drugs

A single injection of saline (0.9% NaCl) or ketamine (30 mg kg⁻¹)(Ketaset III, Ketamine HCl injection, Fort Dodge Animal Health, FortDodge, Iowa) was administered once at 8 weeks of age as previouslydescribed (Brachman et al, 2016; McGowan et al, 2017). Ketamine wasprepared in physiological saline and all injections were administeredintraperitoneally (i.p.) in volumes of 0.1 cc per 10 mg body weight.

Contextual Fear Conditioning (CFC)

A 3-shock CFC paradigm was administered as previously described (Dennyet al, 2014; Drew et al, 2010; McGowan et al, 2017). Mice were placed inthe conditioning chamber and received 3 shocks 180, 240, and 300 s later(2 s, 0.75 mA) and were removed 15 s following the last shock. Forcontext re-exposure, mice were placed back into the context for 180 s.The no-shock control group underwent the same protocol, but did notreceive 3 shocks during the first exposure. All sessions were scored forfreezing using FreezeView2(http://actimetrics.com/downloads/freezeframe/). All behavioralstatistical analyses are included in Table 1.

Brain Extraction

Mice were sacrificed via cervical dislocation 2 h following CFCre-exposure. Brains were extracted and frozen at −20° C. for 30 sec.Brains were cut using a brain matrix slicer (Cat. # BSMAS001-1, ZivicInstruments, Pittsburgh, Pa., USA). Bilateral punches were then excisedfrom the PFC using a brain punch tissue set (Cat. #57401, Stoelting,Wood Dale, Ill.) according to the coordinates of Paxinos and Franklin(2001) in a glass dish containing ice. Left and right punches werestored separately. The HPC was manually dissected and left and righthemispheres were stored separately. Each sample was weighed. The weightsof all samples are included in Table 1. All tissue samples were thenstored at −80° C. until metabolomic analysis.

Blood Plasma Collection

Trunk blood was collected immediately following decapitation and spundown (10,000 rpm) in Eppendorf tubes pre-coated with 5 l 0.5 M EDTA toobtain plasma. Samples were transferred to new tubes and stored at −80°C. until analysis. All tissue samples were then stored at −80° C. untilanalysis.

Preparation of Brain Tissue

Frozen brain tissue was transferred to a homogenization tube containingceramic beads (Omni International, Kennesaw, Ga.). A volume of ice-cold80% MeOH was added to obtain a 20 mg/ml solution to each of the tubes.The tubes were snap frozen in liquid nitrogen during processing toensure the tissue was kept cold. After a brief thawing period, the tubeswere then transferred to a Bead Ruptor Homogenizer (Omni International,Kennesaw, Ga.). Tissue was homogenized for 2 cycles of 45 seconds, witha 15 second rest period in between. Samples were sonicated for 10minutes at room temperature (RT) and then centrifuged at 14,000 rpm for10 minutes at 4° C. All of the supernatant was removed, transferred toan Eppendorf tube, and evaporated to dryness overnight using acentrifugal evaporator. Once dry, the dried lysate was stored at −80° C.until analysis.

Preparation of Plasma

Plasma was stored at −80° C. until extraction. The samples were thawedat RT for 10 minutes. Thirty μl were removed and were added to 270 μl ofice-cold 80% MeOH. The lysate was then vortexed and spun in a centrifugeat 14,000 rpm for 10 minutes at 4° C. One hundred μl of supernatant weretaken and transferred to a fresh Eppendorf tube and evaporated todryness overnight using a centrifugal evaporator. Once dry, the lysatewas stored at −80° C. until analysis.

Metabolomics Analysis

For untargeted metabolomics analysis, brain samples were reconstitutedin 2:1:1 acetonitrile:MeOH:H₂O, to yield a concentration of 200 mg/ml.Plasma samples were reconstituted in the same solvent, at a 30 μlresuspension volume. Both were spun at 14,000 rpm for 10 minutes at 4°C. to remove excess debris before analysis. Chromatography was performedusing an Agilent 1290 Infinity UPLC. Ten μl of each sample were injectedonto a ZIC-pHILIC column (EMD Millipore, Billerica, Mass.) withdimensions of 150×4.6 mm, 5 μm. Metabolites were separated using anacetonitrile/H₂O with 20 mM ammonium carbonate (pH 9.2) gradient over a29-minute period. A 10-minute re-equilibration time was carried out inbetween injections. Detection was performed using an Agilent 6550Quadrupole-time-of-flight (QToF) mass spectrometer, operated in bothnegative and positive modes. Full scan MS data was collected from m/z70-1000 and metabolites were identified in an untargeted manner bylooking within 10 ppm of the expected m/z values. Real-time masscalibration was performed throughout the duration of sample analysis.

Plasma samples were also subjected to a separate analysis of metabolitesinvolved in neurotransmission. The dried plasma samples werereconstituted in 30 μl of H₂O. Five μl of each sample were injected ontoa Kinetix F5 column (Phenomenex, Torrance, Calif.) with dimensions of100×3 mm, 2.6 am. Metabolites were separated using water with 0.2%formic acid and MeOH gradient over a 12-minute period. Are-equilibration time of 3 minutes was carried out in betweeninjections. Similarly, detection was performed using an Agilent 6550QToF mass spectrometer, operated in both negative and positive modes(FIGS. 6A-6Z, 6AA-6HH, 7A-7K, 8A-8V). Full scan MS data was collectedand metabolites were identified using expected m/z values, as well asconfirmation against purified chemical standards. Data was processedusing a publically available software package, MAVEN(https://www.ncbi.nlm.nih.gov/pubmed/22389014). Area under thechromatographic peak for each metabolite was calculated and exported toassess for differences in metabolite abundances.

Pathway Analysis

For each pathway analysis, the metabolites that were changed in bothhemispheres or in the plasma were entered into MetaboAnalyst 3.0 usingtheir HMDB IDs(http://www.metaboanalyst.ca/faces/Secure/upload/PathUploadView.xhtml).

Heat Map

MATLAB (https://www.mathworks.com/, Mathworks, Natick, Mass.) was usedto transform metabolomics data for the heat maps. The functionNormalizeforHeatMap takes a data input that is in the form of a matrix(size X by Y) and normalizes each row to values between 0 and 1. Thisfunction loops through each row, and at each iteration, the functionfirst takes the minimum of each row and subtracts that from all of thevalues in that row. It then divides each value in that row by themaximum value of that row. The data are accumulated into a new matrixthat contains all of the normalized values.

Statistical Analysis

All data were analyzed using StatView 5.0 software (SAS Institute, Cary,N.C.) or Prism 7.0 (Graphpad Software, Inc., La Jolla, Calif.). Alphawas set to 0.05 for all analyses. In general, the effect of Drug wasanalyzed using an analysis of variance (ANOVA), using repeated measureswhere appropriate. Unpaired t-tests were performed on each metaboliteanalyzed. All statistical tests and p values for metabolomic analysesare listed in Table 1.

Results Prophylactic Ketamine Buffers Conditioned Fear Responses

First, to validate our previously reported effect of prophylacticketamine on CFC, we administered a single dose of saline or ketamine (30mg kg⁻¹) to mice 1 week before a 3-shock CFC paradigm (FIG. 1A).Prophylactic ketamine did not alter freezing behavior during CFCencoding (FIG. 1B). As we previously reported (McGowan et al., 2017),during context re-exposure, prophylactic ketamine attenuated the fearresponse (FIG. 1C). In another group of mice, we administered a singledose of saline or ketamine (30 mg kg⁻¹) to mice 1 week before beingexposed to the CFC context, with no shocks (FIG. 9A). Prophylacticketamine did not alter freezing behavior during context exposure orre-exposure (FIG. 9B-9C).

Prophylactic Ketamine Significantly Alters Metabolites in the PFC andHPC Following Stress

A number of positive and negative mode metabolites were changed in thePFC (FIGS. 6A-6Z, 6AA-6HH, 7A-7K) and HPC (FIGS. 8A-8V) followingprophylactic ketamine administration and a CFC stressor. Notably, 8metabolites were changed in both hemispheres of the PFC and HPC (FIG.2A, Table 2). A pathway analysis of changed metabolites in the PFCindicated that purine metabolism; phenylalanine, tyrosine, andtryptophan metabolism; and phenylalanine metabolism are mostsignificantly changed in the PFC following prophylactic ketamineadministration and stress (FIG. 2B). A pathway analysis of changedmetabolites in the HPC indicated that as in the PFC, purine metabolismwas also significantly altered (FIG. 2C). However, prophylactic ketaminetreatment before stress also altered alanine, aspartate, and glutamatemetabolism; glutamine and glutamate metabolism; and taurine andhypotaurine metabolism in the HPC (FIG. 2C).

Next, heat maps of the changed metabolites in the PFC and HPC werecreated for the stressed mice (FIGS. 2D-2E). Heat maps were used tovisualize changes following ketamine treatment in the left versus righthemispheres of each brain region. A number of metabolites had alteredexpression following prophylactic ketamine administration when comparedwith prophylactic saline expression.

Prophylactic Ketamine Significantly Alters Purine Metabolism FollowingStress

Of the 8 metabolites changed, 6 metabolites were changed in the samedirection in both hemispheres of the PFC and HPC of prophylacticketamine-treated mice that had undergone stress, and these metaboliteswere involved in purine and pyrimidine biosynthesis (Table 2). We firstanalyzed individual purine metabolites in the PFC and HPC. Purineprecursors and nucleotides were significantly altered in bothhemispheres of the PFC (FIGS. 3A-3G) and HPC (FIGS. 3H-3L).Specifically, inosine metabolites were decreased following prophylacticketamine administration, and the downstream metabolites adenosine andguanosine metabolites were increased. However, these changes were notobserved in a group of mice that received prophylactic ketamine withoutstress (FIGS. 9A-9W).

Prophylactic Ketamine Significantly Alters Pyrimidine MetabolismFollowing Stress

Pyrimidine metabolites were next analyzed in both the PFC (FIGS. 4A-4E)and HPC (FIGS. 4F-4H). Since both the PFC and HPC showed alterations indeoxyuridine diphosphate (dUDP), we performed a regression analysis ondUDP expression and freezing behavior in both the PFC (FIG. 4I) and inthe HPC (FIG. 4J). In the PFC, dUDP expression is positively correlatedwith freezing levels upon context re-exposure in mice administeredprophylactic ketamine prior to stress. These alterations in pyrimidinemetabolism were not observed in mice that received prophylactic ketaminewithout stress (FIGS. 9A-9W).

Prophylactic Ketamine Significantly Alters Purine and PyrimidineMetabolism in Plasma After Stress

We next predicted that the effects of prophylactic ketamine might beapparent in the periphery as well as in the brain. Therefore,metabolomics analysis was next performed on plasma samples of stressedand non-stressed mice. A pathway analysis was first performed of changedmetabolites indicating that purine metabolism, pyrimidine metabolism,the citrate cycle (TCA cycle), and the pentose phosphate pathway weremost significantly changed following prophylactic ketamineadministration and stress (FIG. 5A). A heat map was created of allmetabolites changed in the plasma between the two groups (FIG. 5B).Prophylactic ketamine administration significantly increased purinemetabolites in the plasma (FIGS. 5C-5L). Moreover, with the exception of5,6-dihydrouridine, prophylactic ketamine administration increasedexpression of pyrimidine metabolites in the plasma (FIG. 5M-5Q).Interestingly, when the mice were not administered shocks, no changes inpurines or pyrimidine metabolites were observed, suggesting thatprophylactic ketamine interacts with stress to produce long-termperipheral changes in purine and pyrimidine metabolism (FIGS. 9A-9W).

Amino Acid-Derived Neurotransmitters and Precursors are SignificantlyChanged Following Prophylactic Ketamine and Stress in the PFC and HPC

All amino-acid derived neurotransmitters and their precursors were nextanalyzed for alterations following prophylactic ketamine administration(Table 3). Interestingly, following stress, almost all inhibitoryneurotransmitter metabolites were increased (e.g., alanine,gamma-aminobutyric acid (GABA), taurine) and nearly all excitatoryneurotransmitter metabolites were decreased (e.g., serine, tyrosine, andphenylalanine). The main exception to this observation was glutamicacid, a precursor to GABA. Therefore, it is possible that the increasein glutamic acid is directly related to the increase in GABA. Incontrast, in non-stressed mice, only the inhibitory neurotransmittermetabolite taurine was changed. These data suggest that prophylacticketamine and stress may increase inhibitory tone in the brain followingadministration, resulting in long-lasting protection.

Amino Acid-Derived Neurotransmitters and Precursors are SignificantlyChanged Following Prophylactic Ketamine and Stress in Plasma

As performed on brain tissue, amino-acid derived neurotransmitters andtheir precursors in plasma were next analyzed for alterations followingprophylactic ketamine administration (Table 3). A number of aminoacid-derived neurotransmitters and precursors were altered, but only instressed mice. Interestingly, as in the brain tissue, two excitatoryneurotransmitters serine and glutamic acid were decreased. However,unlike in the brain tissue, GABA, an inhibitory neurotransmitter, wasdecreased. These data suggest that metabolite profiling of plasmasamples may indicate effectiveness of prophylactic treatment.

Discussion

Here, we have shown that a single injection of ketamine before a 3-shockCFC protocol attenuated learned fear and produced long-lasting changesin the metabolite profile of PFC and HPC tissues, as well as of plasma.Interestingly, a single injection of ketamine without stress did notproduce similar long-lasting changes in the PFC, HPC, or plasma,suggesting that prophylactic ketamine may interact with stress to inducelong-lasting metabolic changes, especially in the periphery, that affectbehavior. To our knowledge, this is the first study that identifiesmetabolite alterations and biomarker candidates for prophylacticketamine efficacy in mice.

Purine and pyrimidine metabolism was affected not only in the PFC and inthe HPC, but also in plasma of prophylactic ketamine-treated stressedmice. Genetically determined aberrations in purine and pyrimidinemetabolism have been associated with neurological dysfunction (Jinnah etal, 2013; Kelley and Andersson, 2014; Micheli et al, 2011) andpsychiatric disorders (Astakhova and Asanova, 1985; Cieslak et al,2016). Purine and pyrimidine metabolism has also recently beenimplicated in antidepressant treatment response in mice and humans (Parket al, 2016). In this study, a commonly used selective serotoninreuptake inhibitor (SSRI), paroxetine, was administered to DBA/2J micefor 28 days. Drug non-responder and responder mice were identified, andthe metabolomic profile was assessed in the HPC and plasma. Theirresults suggest that chronic SSRI treatment differentially affectspurine and pyrimidine metabolism, which may explain the heterogeneousantidepressant treatment response, and represents a potentialbiosignature of the disease. Specifically, the authors observeddecreased purine and pyrimidine metabolism in antidepressantnon-responders, and increased metabolism in responders. Here, we havesimilarly assessed efficacy of the rapid-acting antidepressant ketamine,but when administered prior to stress. The effect of prophylacticketamine on purine and pyrimidine metabolism after stress suggests thatseveral mechanisms of successful antidepressant treatment may overlapwith prophylactic responsiveness. Additionally, to further assess howchanges in the metabolome relate to behavioral output, we correlated thefreezing levels of stressed mice with the amount of the pyrimidine dUDPin both hemispheres of the PFC and HPC. We found that dUDP levelspositively correlated with freezing levels upon context re-exposure, butonly in the PFC of mice administered ketamine, suggesting that thealterations in the metabolome induced by ketamine and stress maydirectly influence pathological states.

Prophylactic ketamine increased precursors to inhibitoryneurotransmitters, while decreasing most precursors to excitatoryneurotransmitters in stressed mice. Increasing evidence demonstratesthat MDD is associated with an imbalance of excitatory and inhibitoryneurotransmitters glutamate and GABA (Hasler et al, 2007). Specifically,glutamic acid and GABA dysregulation is a hallmark of MDD pathology(Abdallah et al, 2014; Pehrson and Sanchez, 2015), as well as of PTSDand other panic and social anxiety disorders (Averill et al, 2016;Meyerhoff et al, 2014). Interestingly, in a model of chronic socialdefeat stress (CSDS), mice that exhibited depressive-like behaviorshowed a reduction in glutamate in the GABAergic pathway, suggestingthat glutamatergic disorders may be implicated in susceptibility tostress (Wang et al, 2016). Collectively, these data suggest thatprophylactic ketamine may be increasing stress resilience by regulatingGABA and glutamate neurotransmission and therefore, preventingstress-induced phenotypes.

No previous studies have identified the alterations in the metabolomethat occur upon administering a resilience-enhancing pharmaceutical, asthe current study sought to assess. However, previous studies have beenconducted in regards to ketamine's rapid-acting antidepressant effectsand the resultant metabolomic and proteomic changes. Specifically,adenosine has been shown to mediate antidepressant action of ketamine,and that enhancement of the adenosine A₁ receptors (AIRs) in forebrainneurons of mice produced resilience-enhancing effects (Serchov et al,2015). In general, adenosine has been known to act as ananti-inflammatory agent involved in neuroprotection, and it ishypothesized that imbalanced levels of adenosine-5′-triphosphate (ATP)and adenosine in the extracellular space may lead to neuropsychiatricdiseases (Cieslak et al, 2016). These data are in agreement with ourresults demonstrating that prophylactic ketamine administrationincreases levels of adenosine monophosphate (AMP) and ATP in bothhemispheres of the PFC and HPC, and in the plasma.

Thyrotropin-releasing hormone (TRH) and TRH-like peptides, which arepotential therapeutic targets for MDD, are also increased in variousbrain regions, including the PFC and HPC, upon ketamine administration(Pekary et al, 2015). It has also been demonstrated that in as rapidlyas 2 hours post-ketamine injection, hippocampal pathways includingglycolysis, pentose phosphate pathway, and citrate cycle are impacted(Weckmann et al, 2014). Importantly, the pentose phosphate pathway isknown to be important for purine formation. Here, we show that riboseand ribulose phosphate, pentose phosphate pathway intermediates, aresignificantly changed in plasma, which is in agreement with Weckmann etal.'s study demonstrating the role of ketamine on the pentose phosphatepathway and purine formation. Thus, there may be a cascade of changesoccurring after ketamine administration that are activated duringstress, which could explain its long-lasting resilience-enhancingeffects, such as changes in the pentose phosphate pathway that lead tochanges in purine formation.

The use of metabolite supplements in the treatment of depression hasbeen attempted, as metabolic syndromes have been associated with MDD(Marazziti et al, 2014; Martinac et al, 2014) and PTSD (Bartoli et al,2013). Interestingly, one clinical study found that up to two-thirds ofTRD patients may demonstrate at least one metabolic abnormality (Pan etal, 2017). Systemic folate deficiency has been of particular relevanceto this study, as cerebral folate deficiency (CFD) was the most commonin the TRD patients. Typically, CFD syndromes present as developmentaland psychiatric disorders (Ramaekers et al, 2013; Serchov et al, 2015).Folate, a water-soluble B-vitamin, is involved in nearly 100 metabolicreactions including the purine synthetic pathway (Pan and Vockley,2013), and is necessary for the biosynthesis of monoamineneurotransmitters serotonin, epinephrine, and dopamine (Serchov et al,2015). Low folate levels have been associated with reduced response toantidepressants, and have been correlated with MDD symptomology; thus,folate supplements such as folic acid or methylfolate have beenprescribed to MDD patients (Nelson, 2012). The influence of folate onmonoamine neurotransmitters is reflected in our data, as folate changesmay have influenced changes we observed in neurotransmitters such astyrosine and phenylalanine in the brain, which are precursors todopamine, norepinephrine, and epinephrine.

The identification of specific metabolite markers associated withpsychiatric disease using metabolomics platforms has only been recentlypursued. There are an emerging series of studies assessing urinary andperipheral blood metabolite biomarkers to aid in diagnosis of MDD (Leeet al, 2016; Redei and Mehta, 2015; Zheng et al, 2013a; Zheng et al,2016; Zheng et al, 2013b). Prior to metabolomics studies, researchfocused on identifying genomic markers of MDD, but efforts have not yetyielded diagnostic and treatment biomarkers in the genome (Miller andO'Callaghan, 2013). Interestingly, we found that prophylactic ketaminesignificantly altered metabolites in both the brain and the plasma afterstress. Importantly, prophylactic ketamine alone does not induce thesame changes in metabolites, but rather, these changes are triggeredonly after the experience of a stressor. This is noteworthy, as itsuggests that there are peripheral changes with prophylactic treatmentand stress that may be identified in the clinic.

Overall, the present study may lead to novel insights on how ketaminetreatment prior to a stressor may alter metabolic pathways that areinvolved in neurotransmission, and how these pathways are implicated inmood disorders. These data suggest that a single dose of ketamineinduces a cascade of effects following a stressor that may underlie itslong-lasting resilience against stress-induced disorders. These resultsmay elucidate potential biological processes in the brain and plasmathat are critical for maintaining resilience against stressors.

Table 1. Statistical analysis summary for CFC data, weights of HPC andPFC samples used for analyses, and statistical analysis summary formetabolomics data.

Table 2. Prophylactic ketamine prior to CFC stress results in alteredpurine and pyrimidine metabolism. Of the 8 metabolites changed followingprophylactic ketamine and CFC stress, 6 metabolites were changed in thesame direction in both hemispheres of the PFC and HPC and thesemetabolites were primarily involved in purine and pyrimidinebiosynthesis.

Table 3. Amino acid-derived neurotransmitters and precursors aresignificantly changed following prophylactic ketamine and CFC stress inthe PFC, HPC, and plasma. Amino-acid derived neurotransmitters and theirprecursors were next analyzed for alterations following prophylacticketamine administration and CFC stress. Interestingly, in the PFC andHPC almost all inhibitory neurotransmitter metabolites were increased(e.g. alanine, gamma-aminobutyric acid (GABA), taurine) and nearly allexcitatory neurotransmitter metabolites were decreased (e.g. serine,tryosine, and phenylalanine). The main exception to this observation wasglutamic acid, a precursor to GABA. Therefore, it is possible that theincrease in glutamic acid is directly related to the increase in GABA.These data suggest that prophylactic ketamine may increase inhibitorytone in the brain following administration, resulting in long-lastingprotection. As performed on brain tissue, metabolomic profiling wasperformed on plasma samples. A number of amino acid-derivedneurotransmitters and precursors were altered following prophylacticketamine and CFC stress. Interestingly, as in the brain tissue, twoexcitatory neurotransmitters serine and glutamic acid were decreased.However, unlike in the brain tissue, GABA, an inhibitoryneurotransmitter, was decreased. These data suggest that metaboliteprofiles of plasma samples may indicate effectiveness of prophylactictreatment.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, and publications are cited throughout thisapplication, the disclosures of which, particularly, including alldisclosed chemical structures, are incorporated herein by reference.Citation of the above publications or documents is not intended as anadmission that any of the foregoing is pertinent prior art, nor does itconstitute any admission as to the contents or date of thesepublications or documents. All references cited herein are incorporatedby reference to the same extent as if each individual publication,patent application, or patent, was specifically and individuallyindicated to be incorporated by reference.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

TABLE 1 Behavioral * of Paradigm Abbrev Measurement Comparison F freedomp * FIG. Contextual CFC D1 Freezing Drug 3.528 1.68 0.0776 — 1B FearTime 33.644 4.68 <0.0001 *** Conditioning Drug × Time 2.275 4.68 0.0701— (3-shock) CFC D2 Freezing Drug 5.191 1.68 0.0359 * 1C Time 27.495 4.68<0.0001 *** Drug × Time 0.519 4.68 0.5996 — Contextual CFC D1 FreezingDrug 0.679 1.64 0.0422 — S5B Fear Time 3.402 4.64 0.0138 * ConditioningDrug × Time 1.579 4.64 0.2621 — (No shock) CFC D2 Freezing Drug 0.4021.64 0.5353 — S5C Time 4.3 4.64 0.0038 ** Drug × Time 0.528 4.64 0.7159— Left Right Total Left Right Total PFC PFC PFC HPC HPC HPC WeightWeight Weight Weight Weight Weight Mouse ID CFC Drug (g) (g) (g) (g) (g)(g) 1 3-shock Sal 0.0156 0.0132 0.0288 0.0156 0.0146 0.0302 2 3-shockSal 0.0151 0.0160 0.0311 0.0209 0.0190 0.0399 3 3-shock Sal 0.02000.0200 0.0400 0.0107 0.0254 0.0361 4 3-shock Sal 0.0284 0.0223 0.05070.0141 0.0168 0.0309 5 3-shock Sat 0.0221 0.0210 0.0431 0.0162 0.01910.0353 6 3-shock Sat 0.0122 0.0100 0.0222 0.0160 0.0150 0.0310 7 3-shockSal 0.0150 0.0110 0.0260 0.0128 0.0170 0.0298 8 3-shock Sat 0.01400.0134 0.0274 0.0125 0.0145 0.0270 9 3-shock Sal 0.0140 0.0150 0.02900.0157 0.0124 0.0281 10 3-shock Sal 0.0130 0.0120 0.0250 0.0132 0.01150.0247 11 3-shock K 0.0195 0.0205 0.0400 0.0150 0.0155 0.0305 12 3-shockK 0.0270 0.0272 0.0542 0.0219 0.0207 0.0426 13 3-shock K 0.0170 0.01300.0300 0.0140 0.0140 0.0280 14 3-shock K 0.0248 0.0223 0.0471 0.01550.0144 0.0299 15 3-shock K 0.0195 0.0174 0.0369 0.0139 0.0123 0.0262 163-shock K 0.0137 0.0110 0.0247 0.0160 0.0147 0.0307 17 3-shock K 0.01300.0150 0.0280 0.0124 0.0135 0.0259 18 3-shock K 0.0200 0.0200 0.04900.0143 0.0130 0.0273 19 3-shock K 0.0150 0.0150 0.0300 0.0190 0.01590.0349 20 No shock Sal 0.0150 0.0129 0.0279 0.0155 0.0177 0.0332 21 Noshock K 0.0183 0.0213 0.0396 0.0137 0.0133 0.027 22 No shock Sal 0.02050.0181 0.0386 0.0221 0.0139 0.036 23 No shock Sal 0.0152 0.0146 0.02980.017 0.0138 0.0308 24 No shock Sal 0.0208 0.0200 0.0408 0.019 0.0170.036 25 No shock K 0.0180 0.0238 0.0418 0.019 0.015 0.034 26 No shock K0.0166 0.0183 0.0349 0.0178 0.015 0.0328 27 No shock K 0.0250 0.01930.0443 0.01 0.0133 0.0233 28 No shock Sal 0.0197 0.0238 0.0435 0.01440.0153 0.0297 29 No shock Sal 0.0278 0.0227 0.0595 0.0176 0.017 0.034630 No shock Sal 0.0190 0.0242 0.0432 0.0159 0.0134 0.0293 31 Ng shockSal 0.0200 0.0219 0.0419 0.0158 0.0132 0.029 32 No shock Sal 0.01430.0161 0.0304 0.0157 0.0142 0.0299 33 No shock K 0.0236 0.0213 0.04490.0175 0.0157 0.0332 34 No shock K 0.0136 0.0168 0.0394 0.0148 0.01230.0271 35 No shock K 0.0211 0.0167 0.0378 0.0145 0.0137 0.0282 36 Noshock K 0.0137 0.0150 0.0287 0.0157 0.0102 0.0259 37 No shock K 0.16900.0166 0.1856 0.0183 0.0122 0.0305 Stressed Mice Left PFC Right PFCRegion Mode Metabolite p value p value PFC Positive 1-Methylhistidine0.0004 0.0032 11-Hydroxyandrosterone 0.0387 0.6853 13-HOTE 0.0021 0.224319-Norandrosterone 0.0377 0.3451 2-Hexenoylcarnitine 0.9272 0.30882-Methoxyestradiol 0.2216 0.4853 3--5-Tetradecaniencarnitine 0.92170.0457 3-5-Diiodothyronine 0.5416 0.1165 3-Aminoisobutanoic-acid 0.00000.0000 3-Methylcrotonylglycine 0.0212 0.0007 3-Methylhistidine 0.00230.0091 3-Methyluridine 0.1794 0.2124 5--Methylthioadenosine 0.00840.1503 5-6-Dihydrouridine 0.6656 0.0929 5-Methylcytidine 0.6431 0.30728-Hydroxyguanine 0.1223 0.0251 9-12-Hexadecadienoylcarnitine 0.21050.0618 9-Decenoylcarnitine 0.4361 0.4361 Acetoacetic-acid 0.0000 0.0038Adenosine-triphosphate 0.0291 0.1557 Alanyl-Proline 0.0025 0.0180Aminoadipic-acid 0.4701 0.5719 Argininosuccinic-acid 0.0274 0.0424Asymmetric-dimethylarginine 0.0050 0.0363 Carnosine 0.0406 0.0337Citrulline 0.0019 0.0050 Cyclic-AMP 0.3953 0.4846 Cystathionine-ketimine0.0310 0.2109 Cytidine 0.6916 0.6714 D-Serine 0.0011 0.0092 D-Tryptophan0.0056 0.0129 Deoxycytidine 0.0165 0.0477 Deoxyuridine 0.0017 0.5072Dihydrothymine 0.0054 0.0095 Dodecanoylcarnitine 0.1678 0.0547 dUDP0.0002 0.0003 Erythronic-acid 0.0096 0.0077 Estradiol 0.0027 0.1554Glucosylgalactosyl-hydroxylsine 0.0050 0.0123 Glycine 0.0136 0.0061Glycylproline 0.0028 0.0100 Guanosine-diphosphate 0.6691 0.3997Guanosine-monophosphate 0.3745 0.0330 Guanosine-triphosphate 0.04330.0544 Hexanoylcarnitine 0.6060 0.0104 Homo-L-arginine 0.0485 0.0871Homocarnosine 0.0069 0.0309 Hypoxanthine 0.0188 0.0096 IMP 0.0031 0.0218Inosine 0.0127 0.0086 Isobutyryl-L-carnitine 0.1272 0.0332 Isoleucine0.0027 0.0105 Isovalerylcarnitine 0.5210 0.2595 Isovalerylglucuronide0.0302 0.0336 Ketoprofen-glucuronide 0.0018 0.0019 L-Allothreonine0.0023 0.0024 L-Cystathionine 0.2210 0.6509 L-Cysteinylglycine-disulfide0.0001 0.0010 L-Homoserine 0.0021 0.0289 L-Octanoylcarnitine 0.53900.0150 L-Tyrosine 0.0022 0.0059 Leucine 0.0000 0.0046 Malonylcarnitine0.0091 0.2928 MET-ENKEPHELIN 0.1336 0.0035 methionine 0.0153 0.0830N--Phosphoguanidinoethyl methyl phosphate 0.2092 0.1060N-Acetyl-L-methionine 0.0325 0.0206 N-Acetylcystathionine 0.0758 0.0198N-Acetylglutamine 0.0154 0.1573 N-Acetylneuramino-acid 0.0045 0.2486N-acetyltryptophan 0.0206 0.1415 N-Acetylvanilanine 0.1695 0.1871N-Formyl-L-methionine 0.7312 0.0085 N4-Acetylaminobutanal 0.0045 0.0518NAD 0.2233 0.0431 NAD+ 0.2640 0.0482 NADH 0.0171 0.0150 Nicotinamide0.0018 0.0597 Nonanoylcarnitine 0.0013 0.0198 p-Cresol-glucuronide0.1964 0.1862 Phenylalanine 0.0064 0.0337 Phenylpyruvic-acid 0.00110.0031 Pipecolic-acid 0.0143 0.0630 Progesterone 0.1866 0.6845Proline-betaine 0.1691 0.4450 Prolylhydroxyproline 0.0577 0.0509S-adenosyl-L-homoCysteine 0.0246 0.1132 S-Adenosylmethionine 0.11360.0061 Suberylglycine 0.1818 0.1991 Succinyladenosine 0.0941 0.0405Taurocholic-acid 0.0033 0.0510 Taurodeoxycholic-acid 0.0000 0.0006Testosterone 0.0698 0.0089 Thiamine-monophosphate 0.0128 0.0223Thyroxine 0.1623 0.1869 trans-2-Dodecenoylcarnitine 0.4636 0.0027Trimethylamine-N-oxide 0.4867 0.3878 Uracil 0.0840 0.0423 Urolithin-C0.0410 0.7105 Xanthine 0.0392 0.0515 Xanthurenic-acid 0.6185 0.0141Negative (S)-Lactaldehyde 0.0553 0.0019 11b-Hydroxyprogesterone 0.00830.3228 12-13-DHOME 0.0068 0.4807 1-Methylxanthine 0.6021 0.00002-Arachidonylglcerol 0.0127 0.3843 2-Haxenoylcarnitine 0.0308 0.56442-Hydroxyadipic-acid 0.2927 0.0028 2-Phenylpropionate 0.0569 0.62443-Chlorotyrosine 0.0009 0.2610 3-Detrydrocamitine 0.0834 0.95973-Hydroxydodecanoic-acid 0.0022 0.6950 3-Hydroxytetradecanedioic-acid0.0037 0.9384 3-Sullinoalanine 0.5418 0.6465 5-6-Dihydrouridine 0.60660.0001 5-Methylcytidine 0.1470 0.0123 5-Tetradecenoic-acid 0.0467 0.88976-Hydroxydaidzein 0.0013 0.7263 Adenine 0.3341 0.0044 Adenosine 0.30860.0045 Adenosine-monophosphate 0.0318 0.0011 Adenosine-triphosphate0.0142 0.0164 ADP 0.5191 0.0143 ADP-Glucose 0.8177 0.0002Aminoadipic-acid 0.1931 0.0000 Butyrylcarnitine 0.0027 0.8076 CDP 0.77950.0271 Cyclic-AMP 0.7048 0.0044 Cytidine 0.1892 0.0205Cytidine-monophosphate 0.9227 0.0051 Cytosine 0.2507 0.0030 D-Alanine0.0178 0.0000 Dihydrothymine 0.0200 0.0001 Dihydroxyacetone-phosphate0.4940 0.0052 D-Lysine 0.6999 0.0077 Dodecanedioic-acid 0.0021 0.9179Dodecanoic-acid 0.0447 0.8702 D-Serine 0.1125 0.0000 D-Tryptophan 0.02200.2810 Enkephalin-L 0.1345 0.0007 FAD 0.3991 0.0503Fructose-1-6-bisphosphate 0.1661 0.0415 Gamma-Aminobutyric-acid 0.52520.0000 Gamma-Glutamyl-Glutamine 0.0651 0.3903 Glucosamine-phosphate0.0037 0.0044 glutathione 0.4697 0.0422 Glyceric-acid 0.6690 0.0000Glycerol-3-phosphate 0.4140 0.0071 GMP 0.2672 0.0225Guanosine-diphosphate 0.0445 0.0004 Guanosine-triphosphate 0.0194 0.0041Hexanoylcarnitine 0.0339 0.8636 Homocarnosine 0.8750 0.0000Homocitrulline 0.0019 0.3921 Hydroxyhexanoycarnitine 0.0037 0.2976Hydroxyoctanoic-acid 0.0048 0.6490 Hydroxypyruvic-acid 0.0214 0.0003 IMP0.0168 0.6032 Inosine 0.0416 0.4426 Isovaleric-acid 0.5087 0.0045L-2-Hydroxyglutaric-acid 0.3218 0.0096 L-3-Phenyllactic-acid 0.86000.0032 L-Acetylcarnitine 0.0060 0.7490 L-Carnitine 0.2011 0.0038L-Glutamic-acid 0.3732 0.0000 Linoleic-acid 0.0115 0.5766 L-Lactic-acid0.0348 0.0081 L-Malic-acid 0.0373 0.3364 L-Tyrosine 0.0077 0.7414Malonate 0.0214 0.0003 Methionine 0.2286 0.0330 Methylmalonic-acid0.0426 0.0032 N-Acetylglutamine 0.0694 0.6250 NAD+ 0.0739 0.0043 NADH0.0125 0.0021 N-Formyl-L-methionine 0.0092 0.0496 Nicotinamide-ribotide0.1333 0.0083 Octulose-phosphate 0.0093 0.0043 Oleic-acid 0.0317 0.1234O-Phosphoethandamine 0.3872 0.0000 Oxidized-glutathione 0.1284 0.0001Oxoadipic-acid 0.0041 0.1049 Oxoglutaric-acid 0.0274 0.0001Palmitic-acid 0.0261 0.5972 Palmitoleic-acid 0.0024 0.6231p-Cresol-sulfate 0.0018 0.4236 Phenylalanine 0.1502 0.0007Phenylpropionylglycine 0.0071 0.3014 Phosphoenolpyruvic-acid 0.04040.1528 Propionylcarnitine 0.0018 0.2549 Propionylglycine 0.2466 0.0000Ribothymidine 0.0000 0.1674 Sarcosine 0.1092 0.0251Sphingosine-1-phosphate 0.9527 0.0042 Stearic-acid 0.0727 0.2743 Taurine0.8738 0.0000 Testosterone-sulfate 0.0009 0.9284 Tetradecanedioic-acid0.0035 0.7318 Thiamine-pyrophosphate 0.2380 0.0001 Thymine 0.0016 0.0733Uracil 0.2300 0.5986 Uric-acid 0.5679 0.0024 Uridine 0.1539 0.0332Uridine-diphosphate-glucose 0.3417 0.0100Uridine-diphosphate-N-acetylglucosamine 0.0536 0.0170Uridine-triphosphate 0.0085 0.0202 Urolithin-C 0.0011 0.2275Valeric-acid 0.2847 0.0003 Valerylcarnitine 0.0133 0.9297 Xanthine0.0661 0.6403 Xanthosine-5-phosphate 0.3232 0.0042 Left HPC Right HPCRegion Mode Metabolite p value p value HPC Positive 1-Methylhistidine0.0416 0.1744 11-Hydroxyandrosterone 0.5556 0.5952 13-HOTE 0.0421 0.024219-Norandrosterone 0.8228 0.6511 2-Hexenoylcarnitine 0.6606 0.96632-Methoxyestradiol 0.4757 0.9931 3--5-Tetradecadiencarnitine 0.05880.1658 3-5-Diiodothyronine 0.3726 0.6775 3-Aminoisobutanoic-acid 0.03450.0605 3-Methylcrotonylglycine 0.0648 0.7030 3-Methylhistidine 0.01270.0598 3-Methyluridine 0.2461 0.8378 5--Methylthioadenosine 0.68370.0074 5-6-Dihydrouridine 0.0022 0.0030 5-Methylcytidine 0.0540 0.36788-Hydroxyguanine 0.0152 0.0405 9-12-Hexadecadienoylcarnitine 0.09840.7821 9-Decenoylcarnitine 0.0880 0.1751 Acetoacetic-acid 0.7095 0.8305Adenosine-triphosphate 0.0704 0.1266 Alanyl-Proline 0.0296 0.1327Aminoadipic-acid 0.0690 0.2016 Argininosuccinic-acid 0.3259 0.4922Asymmetric-dimethylarginine 0.8378 0.6567 Carnosine 0.6346 0.8656Citrulline 0.1826 0.5655 Cyclic-AMP 0.0875 0.1405 Cystathionine-ketimine0.0443 0.8138 Cytidine 0.1426 0.2717 D-Serine 0.1869 0.8959 D-Tryptophan0.2462 0.4534 Deoxycytidine 0.4804 0.3590 Deoxyuridine 0.2250 0.6074Dihydrothymine 0.0808 0.1165 Dodecanoylcarnitine 0.4180 0.8433 dUDP0.0029 0.0051 Erythronic-acid 0.0185 0.1153 Estradiol 0.4064 0.9947Glucosylgalactosyl-hydroxylysine 0.2679 0.1711 Glycine 0.2668 0.8485Glycylproline 0.0892 0.2478 Guanosine-diphosphate 0.0363 0.0685Guanosine-monophosphate 0.6696 0.2641 Guanosine-triphosphate 0.03190.0736 Hexanoylcarnitine 0.0342 0.0942 Homo-L-arginine 0.1317 0.4405Homocarnosine 0.9057 0.3667 Hypoxanthine 0.0287 0.1542 IMP 0.0134 0.0204Inosine 0.0146 0.0621 Isobutyryl-L-carnitine 0.1058 0.0577 Isoleucine0.1750 0.4787 Isovalerylcarnitine 0.1611 0.3156 Isovalerylglucuronide0.2864 0.8821 Ketoprofen-glucuronide 0.0010 0.0564 L-Allothreonine0.1602 0.8601 L-Cystathionine 0.0878 0.0344 L-Cysteinylglycine-disulfide0.3245 0.4862 L-Homoserine 0.8507 0.1369 L-Octanoylcarnitine 0.00140.0234 L-Tyrosine 0.0522 0.2079 Leucine 0.1488 0.2082 Malonylcarnitine0.9241 0.8249 MET-ENKEPHELIN 0.0795 0.7499 Methionine 0.7775 0.8700N--Phosphoguanidinoethyl methyl phosphate 0.2701 0.4484N-Acetyl-L-methionine 0.3115 0.9274 N-Acetylcystathionine 0.2693 0.1513N-Acetylglutamine 0.5036 0.9774 N-Acetylneuraminic-acid 0.6110 0.5381N-acetyltryptophan 0.0594 0.1616 N-Acetylvanilanine 0.3890 0.4706N-Formyl-L-methionine 0.0885 0.7685 N4-Acetylaminobutanal 0.6462 0.6132NAD 0.0278 0.0430 NAD+ 0.0301 0.0461 NADH 0.0075 0.0877 Nicotinamide0.0743 0.0159 Nonanylcarnitine 0.1313 0.0383 p-Cresol-glucuronide 0.63770.8879 Phenylalanine 0.7497 0.8954 Phenylpyruvic-acid 0.0685 0.2399Pipecolic-acid 0.9596 0.7845 Progesterone 0.2279 0.3776 Proline-betaine0.2189 0.4441 Prolylhydroxyproline 0.2644 0.6170S-adenosyl-L-homoCysteine 0.2590 0.5549 S-Adenosylmethionine 0.10130.0063 Suberylglycine 0.8441 0.1883 Succinyladenosine 0.1831 0.4121Taurocholic-acid 0.7990 0.7520 Taurodeoxycholic-acid 0.5248 0.1932Testosterone 0.0018 0.0128 Thiamine-monophosphate 0.0608 0.0215Thyroxine 0.5634 0.4695 trans-2-Dodecenoylcarnitine 0.0218 0.1847Trimethylamine-N-oxide 0.4533 0.0275 Uracil 0.0917 0.4630 Urolithin-C0.5196 0.2006 Xanthine 0.0628 0.3702 Xanthurenic-acid 0.3880 0.9391Negative (S)-Lactaldehyde 0.0064 0.0042 11b-Hydroxyprogesterone 0.34880.5851 12-13-DHOME 0.4395 0.0008 1-Methylxanthine 0.3016 0.23752-Arachidonylglycerol 0.2195 0.0671 2-Hexenoylcarnitine 0.2634 0.00042-Hydroxyadipic-acid 0.8925 0.0000 2-Phenylpropionate 0.7219 0.32783-Chlorotyrosine 0.7517 0.0182 3-Dehydrocarnitine 0.6270 0.00663-Hydroxydodecanoic-acid 0.7606 0.0327 3-Hydroxytetradecanedioic-acid0.2857 0.0021 3-Sulfinoalanine 0.0589 0.0003 5-6-Dihydrouridine 0.15740.0085 5-Methyloytidine 0.0284 0.1033 5-Tetradecenoic-acid 0.1464 0.36736-Hydroxydaidzein 0.9145 0.0097 Adenine 0.0130 0.0550 Adenosine 0.01280.0612 Adenosine-monophosphate 0.0150 0.0258 Adenosine-triphosphate0.0747 0.0670 ADP 0.0247 0.0051 ADP-Glucose 0.2876 0.0094Aminoadipic-acid 0.1148 0.0009 Butyrylcarnitine 0.4150 0.0148 CDP 0.03270.0073 Cyclic-AMP 0.0554 0.0227 Cytidine 0.0969 0.0899Cytidine-monophosphate 0.7747 0.1819 Cytosine 0.0670 0.0331 D-Alanine0.0025 0.0000 Dihydrothymine 0.4422 0.0076 Dihydroxyacetone-phosphate0.3423 0.0431 D-Lysine 0.1333 0.0004 Dodecanedioic-acid 0.3113 0.0040Dodecanoic-acid 0.4542 0.9220 D-Serine 0.2479 0.0008 D-Tryptophan 0.40390.1403 Enkephalin-L 0.0776 0.0319 FAD 0.6285 0.0449Fructose-1-6-bisphosphate 0.5368 0.1262 Gamma-Aminobutyric-acid 0.00420.0001 Gamma-Glutamyl-Glutamine 0.1732 0.3545 Glucosamine-phosphate0.9447 0.0011 glutathione 0.4302 0.2151 Glyceric-acid 0.9877 0.0015Glycerol-3-phosphate 0.0328 0.0855 GMP 0.5653 0.2310Guanosine-diphosphate 0.0169 0.0099 Guanosine-triphosphate 0.0475 0.0131Hexanoylcarnitine 0.7981 0.0055 Homocarnosine 0.2542 0.2154Hornocitrulline 0.0975 0.0036 Hydroxyhexanoycarnitine 0.5054 0.9992Hydroxyoctanoic-acid 0.1974 0.0429 Hydroxypyruvic-acid 0.8104 0.0068 IMP0.0172 0.1582 Inosine 0.0899 0.9878 Isovaleric-acid 0.2710 0.2508L-2-Hydroxyglutaric-acid 0.0346 0.0060 L-3-Phenyllactic-acid 0.27680.0227 L-Acetylcarnitine 0.4143 0.5135 L-Carnitine 0.4812 0.1800L-Glutamic-acid 0.0100 0.0001 Linoleic-acid 0.1818 0.8592 L-Lactic-acid0.1026 0.0003 L-Malic-acid 0.6960 0.0005 L-Tyrosine 0.0993 0.5132Malonate 0.8104 0.0068 Methionine 0.8526 0.0036 Methylmalonic-acid0.0070 0.0051 N-Acetylglutamine 0.1734 0.0489 NAD+ 0.0360 0.0356 NADH0.0092 0.0830 N-Formyl-L-methionine 0.4225 0.0019 Nicotinamide-ribotide0.3209 0.0089 Octulose-phosphate 0.0722 0.0685 Oleic-acid 0.3155 0.0284O-Phosphoethanolamine 0.0097 0.0043 Oxidized-glutathione 0.1527 0.0094Oxoadipic-acid 0.7109 0.0104 Oxoglutaric-acid 0.4281 0.0126Palmitic-acid 0.2218 0.0018 Palmitoleic-acid 0.2921 0.9979p-Cresol-sulfate 0.6283 0.1339 Phenylalanine 0.9799 0.0041Phenylpropionylglycine 0.5040 0.0057 Phosphoenolpyruvic-acid 0.09740.0335 Propionylcarnitine 0.4493 0.0091 Propionylglycine 0.2793 0.0001Ribothymidine 0.0099 0.9998 Sarcosine 0.4234 0.0000Sphingosine-1-phosphate 0.4054 0.0005 Stearic-acid 0.0657 0.0000 Taurine0.0022 0.0000 Testosterone-sulfate 0.2940 0.0078 Tetradecanedioic-acid0.1047 0.0024 Thiamine-pyrophosphate 0.2377 0.0688 Thymine 0.4149 0.9098Uracil 0.2814 0.5391 Uric-acid 0.5572 0.0086 Uridine 0.7700 0.1794Uridine-diphosphate-glucose 0.0561 0.0606Uridine-diphosphate-N-acetylglucosamine 0.2481 0.3630Uridine-triphosphate 0.1983 0.1072 Urolithin-C 0.8878 0.0469Valeric-acid 0.1203 0.0099 Valerylcarnitine 0.2109 0.0014 Xanthine0.0667 0.8798 Xanthosine-5-phosphate 0.0218 0.0671 Region ModeMatabolite p value Plasma N/A (R)-lipoic acid 0.0298 — 2-Methylcitricacid 0.0003 — 3-Methyluridine 0.0043 — 3-Phosphoglyceric acid 0.0001 —4-Methylcatechol 0.0002 — 5-6-Dihydrouridine 0.0083 —5-Hydroxy-L-tryptophan 0.0086 — Adenine 0.0031 — Adenosine monophosphate0.0057 — Adenosine triphosphate 0.0103 — Allantoin 0.0301 — Argininicacid 0.0470 — Cysteineglutathione disulfide 0.0041 — Cytidinemonophosphate 0.0007 — D-Aspartic acid 0.0032 — Dihydrothymine 0.0058 —DL-2-Aminooctanoic acid 0.0003 — D-Ribose 5-phosphate 0.0004 —D-Ribulose 5-phosphate 0.0005 — D-Serine 0.0178 — Erythronic acid 0.0469— Gamma-Aminobutryic acid 0.0194 — Glycerol 3-phosphate 0.0213 —Guanosine diphosphate 0.0236 — Guanosine monophosphate 0.0103 —Homocarnosine 0.0072 — Hypotaurine 0.0527 — Hypoxanthine 0.0545 —Inosine 0.0511 — L-Cystathionine 0.0044 — L-Glutamic acid 0.0223 —L-Malic acid 0.0005 — Malonylcarnitine 0.0020 — Methylmalonylcarnitine0.0045 — N-Acetylglutamic acid 0.0084 — N-Acetyl-L-tyrosine 0.0438 —N-Acetylneuraminic acid 0.0399 — Nicotinamide N-oxide 0.0084 — Octulosephosphate 0.0275 — O-Phosphoethanolamine 0.9012 — Oxidized glutathione0.0260 — Pantothenate 0.0539 — Phenylpyruvic acid 0.0107 —Phosphoenolpyruvic acid 0.0390 — Phosphoribosyl pyrophosphate 0.0008 —S-Adenosylmethionine 0.0476 — Thiamine pyrophosphate 0.0039 — Threonicacid 0.0206 — trans-Aconitic acid 0.0081 — Tryptophanamide 0.0002 —Uridine 5--diphosphate 0.0002 — Uridine 5--monophosphate 0.0010 —Uridine diphosphate glucose 0.0017 — Non-Stressed Mice Purine or LeftPFC Right PFC Region Pyrimidine Metabolite p value p value PFC PurinesAdenosine monophosphate 0.2928 0.4380 Adenosine triphosphate 0.64850.5829 Guanosine diphosphate 0.8918 0.3221 Guanosine triphosphate 0.05000.0689 Hypoxanthine 0.0339 0.9303 Inosine 0.0458 0.6518 Inosinemonophosphate 0.4642 0.1102 Pyrimidines dUDP 0.4351 0.08533-Aminoisobutanoic acid 0.0029 0.2381 Dihydrothymine 0.7843 0.7012Deoxycytidine 0.9995 0.2264 Uridine triphosphate 0.6823 0.1330 Purine orLeft HPC Right HPC Region Pyrimidine Metabolite p value p value HPCPurines Adenosine-diphosphate 0.2328 0.9157 Adenosine-monophosphate0.9061 0.6704 Guanosine-diphosphate 0.9460 0.2914 Guanosine-triphosphate0.2320 0.1292 Inosine-monophosphate 0.9894 0.0222 PyrimidinesDeoxyurbine-diphosphate 0.7517 0.0049 5-6-Dihydrouridine 0.4247 0.4329Cytidine-diphosphate 0.5557 0.4807 Purine or Region PyrimidineMetabolite p value Plasma Purines Adenine 0.5871 — Adenosinemonophosphate 0.8697 — Adenosine triphosphate 0.7528 —D-Ribose-5-phosphate 0.6598 — D-Ribulose-5-phosphate 0.1537 — Guanosinemonophosphate 0.2434 — Guanosine diphosphate 0.4332 — Hypoxanthine0.4893 — Inosine 0.1620 — Phosphoribosyl pyrophosphate 0.1417 —Pyrimidines 5-6-Dihydrouridine 0.6810 — Cytidine monophosphate 0.8047 —Dihydrothymine 0.4706 — Uridine 5′-monophosphate 0.1285 — Uridine5′-diphosphate 0.3336 —

TABLE 2 Common Name Abbreviation Class Adenosine monophosphate AMPPurine ribonucleoside monophosphates Deoxyuridine-diphosphate dUDPPyrimidine 2′-deoxyribonucleoside diphosphates Guanosine diphosphate GDPPurine ribonucleoside diphosphates Guanosine triphosphate GTP Purineribonucleoside triphosphates Inosine monophosphate IMP Purineribonucleoside monophosphates Methylmalonic-acid — Dicarboxylic acidsand derivatives

TABLE 3 Excitatory/ Name Abbreviation Sample Change Group InhibitoryReceptor Notes D-Alanine Ala HPC Increase CFC Inhibitory Glycinereceptor Has been shown to increase following anti-depressant treatment.D-Serine Ser PFC Decrease CFC Excitatory NDMA receptors and Coagonist atthe glycine glycine receptors site of NDMA receptors. Gamma-aminobuytricGABA HPC Increase CFC Inhibitory GABAA receptors and Low GABA levelshave acid GABAA-p receptors been linked to depression. Glycine Gly PFCDecrease CFC Inhibitory/ NDMA receptors and Often reteased with GABA.Excitatory glycine receptors L-Glutamic acid Glu HPC Increase CFCExcitatory NDMA receptors and Precursor to GABA as well. AMPA receptorsL-Tyrosine Tyr PFC Decrease CFC Excitatory CaSR Precursor toneurotransmitters such as tyrosine, dopamine, norepinephrine, andepinephrine. O-Phosphoethanolamine* PE HPC Increase CFC — — Shows strongstructural similarity to GABA. Co-released with taurine. PhenylalaninePhe PFC Decrease CFC Excitatory CaSR Precursor to neurotransmitters suchas tyrosine, dopamine, norephinephrine, and epinephrine. Taurine Tau HPCIncrease CFC and Inhibitory Glycine receptor Structural resemblance withGABA. context exposure 5-Hydroxy-L-tryptophan 5-HTP Plasma Increase CFC— — Immediate precursor to serotonin (5-HT), which elevates mood.D-Serine Ser Plasma Decrease CFC Excitatory NDMA receptors and Enhancesglutamatergic glycine receptors signalling via NMDARs.Gamma-aminobuytric GABA Plasma Decrease CFC Inhibitory GABAA receptorsand Homeostatic balance of acid GABAA-p receptors GABAergic inhibitionis essential tor controlling exitatory neurotransmission. L-Glutamicacid Glu Plasma Decrease CFC Excitatory NDMA receptors and Precursor toglutamine, AMPA receptors which stabilizes the immune system in times ofstress. N-Acetyl-L-tyrosine NALT Plasma Decrease CFC — — Precursor totyrosine, which converts to norepinephrine (NE) and dopamine (DA),enhancers of mood. O-Phosphoethanolamine* PE Plasma Increase CFC — —Inhibits mitochondrial dysfunction, suggested to be involved in thepathophysiology of affective illnesses.

What is claimed is:
 1. A method for treating a subject for astress-induced affective disorder or stress-induced psychopathology, themethod comprising: (a) administering or having administered aprophylactic treatment to the subject prior to a stressor; (b)determining level of one or more biomarkers in a biological sampleobtained from the subject after step (a) and after the stressor; and (c)comparing the level obtained in step (b) with the level of the one ormore biomarkers in a control sample.
 2. A method for assessing aprophylactic treatment of a subject for a stress-induced affectivedisorder or stress-induced psychopathology, the method comprising: (a)determining level of one or more biomarkers in a biological sampleobtained from the subject after a prophylactic treatment and after astressor; and (b) comparing the level obtained in step (a) with thelevel of the one or more biomarkers in a control sample.
 3. The methodof claims 1 or 2, further comprising maintaining a treatment regime ordetermining the prophylactic treatment as being effective, when thelevel of one or more biomarkers increases or decreases by at least 10%compared to its level in the control sample.
 4. The method of claims 1or 2, further comprising adjusting a treatment regime, when the level ofone or more biomarkers is unchanged, or increases or decreases by lessthan 10%, compared to its level in the control sample.
 5. The method ofclaims 1 or 2, wherein the biomarkers are selected from the groupconsisting of purines, purine metabolites, purine precursors, purinederivatives, pyrimidines, pyrimidine metabolites, pyrimidine precursors,pyrimidine derivatives, nucleotides, nucleotide metabolites, nucleotideprecursors, nucleotide derivatives, neurotransmitters, neurotransmittermetabolites, neurotransmitter precursors, neurotransmitter derivatives,and combinations thereof.
 6. The method of claim 5, wherein theneurotransmitters are inhibitory neurotransmitters or excitatoryneurotransmitters.
 7. The method of claim 5, wherein theneurotransmitters are amino acid-derived neurotransmitters.
 8. Themethod of claims 1 or 2, wherein the biomarkers are selected from thoselisted in FIGS. 5C-5Q.
 9. The method of claims 1 or 2, wherein thebiomarkers are selected from the group consisting of adenine, adenosinemonophosphate (AMP), adenosine triphosphate (ATP), D-ribose 5-phosphate,D-ribulose 5-phosphate, guanosine monophosphate, guanosine diphosphate,hypoxanthine, inosine, phosphoribosyl pyrophosphate, and combinationsthereof.
 10. The method of claims 1 or 2, wherein the biomarkers areselected from the group consisting of cytidine monophosphate,dihydrothymine, uridine 5-monophosphate, and uridine 5-diphosphate,5,6-dihydrouridine, and combinations thereof.
 11. The method of claims 1or 2, wherein the biomarkers are selected from those listed in Table 3.12. The method of claims 1 or 2, wherein the biomarkers are selectedfrom the group consisting of serine, glutamic acid, gamma-aminobuytricacid (GABA), 5-hydroxy-L-tryptophan (5-HTP), O-phosphoethanolamine (PE),N-acetyl-L-tyrosine, and combinations thereof.
 13. The method of claims1 or 2, wherein the prophylactic treatment comprises administering aneffective amount of a pharmaceutic composition comprising ketamine, aketamine analog, or a pharmaceutically acceptable salt, derivative, ormetabolite thereof, to the subject prior to the stressor.
 14. The methodof claims 1 or 2, wherein the prophylactic treatment comprisesadministering an effective amount of a pharmaceutic compositioncomprising an antagonist of the glutamate N-methyl-D-aspartate (NMDA)receptor to a subject prior to a stressor.
 15. The method of claim 14,wherein the antagonist of the NMDA receptor comprises ketamine, aketamine analog, or a pharmaceutically acceptable salt, derivative, ormetabolite thereof.
 16. The method of claims 1 or 2, wherein theprophylactic treatment comprises administering an effective amount of apharmaceutic composition comprising ana-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptoragonist, to a subject prior to a stressor.
 17. The method of claim 16,wherein the AMPA receptor agonist is selected from the group consistingof glutamate, AMPA, 5-fluorowillardiine, domoic acid, quisqualic acid,and (2R,6R)-hydroxynorketamine, CX546, or a pharmaceutically acceptablesalt, derivative, or metabolite thereof.
 18. The method of any of claims13-17, wherein the pharmaceutic composition is administered to thesubject about 48 hours to about 3 weeks prior to a stressor.
 19. Themethod of any of claims 13-17, wherein the pharmaceutic composition isadministered to the subject about 72 hours to about 2 weeks prior to astressor.
 20. The method of any of claims 13-17, wherein thepharmaceutic composition is administered to the subject about 1 weekprior to a stressor.
 21. The method of any of claims 13-17, wherein thepharmaceutic composition is administered to the subject once prior to astressor.
 22. The method of any of claims 13-17, wherein thepharmaceutic composition is administered orally, intravenously,intranasally, or via injection to the subject.
 23. The method of any ofclaims 13-17, wherein the pharmaceutic composition comprisesnorketamine, hydroxyketamines, dehydronorketamine, and/orhydroxynorketamine (HNK).
 24. The method of any of claims 13-17, whereinthe pharmaceutic composition comprises (2R,6R)-HNK and/or (2S,6S)-HNK.25. The method of claim 3, wherein the increase or decrease in the levelof one or more biomarkers is at least 50%.
 26. The method of claim 3,wherein the increase or decrease in the level of the one or morebiomarkers is at least 70%.
 27. The method of claim 3, wherein theincrease or decrease in the level of the one or more biomarkers is atleast 90%.
 28. The method of claim 3, wherein the increase or decreasein the level of the one or more biomarkers ranges from about 30% toabout 3-fold.
 29. The method of claims 1 or 2, wherein the biologicalsample is a plasma, serum, blood or urine sample.
 30. The method ofclaims 1 or 2, wherein the control sample is from a healthy subject or aplurality of healthy subjects.
 31. The method of claims 1 or 2, whereinthe control sample is from a subject who has had a prophylactictreatment without experiencing a stressor, or a plurality of patientswho have had a prophylactic treatment without experiencing a stressor.32. The method of claims 1 or 2, wherein the control sample is from asubject who has experienced a stressor without a prophylactic treatment,or from a plurality of patients who have experienced a stressor withouta prophylactic treatment.
 33. The method of claims 1 or 2, wherein thelevel of the one or more biomarkers is determined by mass spectrometry(MS).
 34. The method of claims 1 or 2, wherein the level of the one ormore biomarkers is determined by chromatography coupled with MS.
 35. Themethod of any of the preceding claim, wherein the stress-inducedaffective disorder comprises major depressive disorder (MDD) and/orposttraumatic stress disorder (PTSD).
 36. The method of any of thepreceding claim, wherein the stress-induced affective disorder isselected from the group consisting of: depressive-like behavior andassociated affective disorders, anhedonic behavior and associatedaffective disorders, anxiety and associated affective disorders,cognitive impairments and deficits and associated disorders, andcombinations thereof.
 37. The method of claims 1 or 2, wherein thestress-induced affective disorder comprises stress-inducedpsychopathology.
 38. The method of claim 37, wherein the stress-inducedpsychopathology comprises depressive and/or anxious behavior.
 39. Themethod of any of the preceding claim, wherein the subject is a mammal.40. The method of any of the preceding claim, wherein the subject is ahuman.
 41. The method of any of claims 13-17, wherein the pharmaceuticalcomposition is administered in a booster series.